HK1149193A

HK1149193A - Combination methods of treating cancer

Info

Publication number: HK1149193A
Application number: HK11103340.8A
Authority: HK
Inventors: G. Bacopoulos Nicholas; H. Chiao Judy; A. Marks Paul; A. Miller Thomas; M. Paradise Carolyn; M. Richon Victoria; A. Rifkind Richard
Original assignee: Sloan-Kettering Institute For Cancer Research; Aton Pharma, Inc.
Priority date: 2003-08-29
Filing date: 2011-04-01
Publication date: 2011-09-30

Description

Methods of combination treatment of cancer

The present application is a divisional application of a patent application having an application date of 2004, 8/12, application No. 200480031561.2 (international application No. PCT/US2004/026161), entitled "method for combined treatment of cancer".

Technical Field

The present invention relates to methods of treating cancer by administering a Histone Deacetylase (HDAC) inhibitor in combination with an anti-cancer agent. The first and second amounts together comprise a therapeutically effective amount.

Background

Cancer is a condition in which a population of cells is, to varying degrees, unresponsive to the control mechanisms that normally determine proliferation and differentiation.

Therapeutic drugs for clinical cancer treatment can be divided into 6 classes: alkylating agents, antibiotic agents, antimetabolite agents, biological agents, hormonal agents, and plant derived agents.

Attempts have also been made to treat Cancer by inducing terminal differentiation of tumor cells (M.B., Roberts, A.B., and Driscoll, J.S. (1985), Cancer: Principles and Practice of Oncology, Hellman, S., Rosenberg, S.A. and DeVita, V.T., Jr eds., 2 nd edition, (J.B.Lippincott, Philadelphia); page 49). Reportedly, in cell culture models, the stimulation of cells by exposure to various stimuli includes: cyclic AMP and tretinoin (Breitman, T.R., Seionick, S.E. and Collins, S.J. (1980) Proc.Natl.Acad.Sci.USA 77: 2936-. There is a great deal of evidence that neoplastic transformation does not require disruption of the differentiation potential of Cancer cells (Sporn et al; Marks, P.A., Sheffery, M. and Rifkind, R.A (1987) Cancer Res.47: 659; Sachs, L. (1978) Nature (Lond.) -274: 535).

There are many examples of tumor cells that do not respond to normal proliferation regulators, and it is clear that expression of their differentiation program is blocked, thereby inducing differentiation and terminating replication. Various drugs can induce various transformed cell lines and primary human tumor transplants to express more differentiation characteristics. These drugs include:

a) polar compounds (Marks et al (1987); friend, c., Scher, w., Holland, j.w., and Sato, T. (1971) proc.natl.acad.sci. (USA) 68: 378-382; tanaka, m., Levy, j., Terada, m., Breslow, r., Rifkind, r.a. and Marks, p.a. (1975) proc.natl.acad.sci. (USA) 72: 1003-1006; reuben, r.c., life, r.l., Breslow, r.r., Rifkind, r.a., and Marks, p.a. (1976) proc.natl.acad.sci. (USA) 73: 862-866);

b) derivatives of vitamin D and tretinoin (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 while 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-;

c) steroid hormones (Lotem, j. and Sachs, L. (1975) int.j. cancer 15: 731-740);

d) growth factor (Sachs, L. (1978) Nature (Lond.) 274: 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 vs 5162); and

g) inhibitors of DNA or RNA synthesis (Schwartz, e.l., and Sartorelli, a.c. (1982) Cancer res.42: 2651-: 2795-; 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 sartorlli, a.c. (1983) Cancer res.43: 2725-; sugano, h., Furusawa, m., Kawaguchi, t., and Ikawa, Y. (1973) bibl.hematol.39: 943-954; ebert, p.s., Wars, i., and Buell, D.N, (1976) cancer res.36: 1809-1813; hayashi, m., Okabe, j., and Hozumi, m. (1979) Gann 70: 235-238).

Histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) belong to the class of drugs that have the ability to induce growth arrest, differentiation and/or apoptosis in tumor cells (Richon, v.m., Webb, y., Merger, r. et al (1996) PNAS 93: 5705-8). These compounds are targeted to become an intrinsic mechanism of malignant cell capacity because they do not appear toxic at doses effective to inhibit 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 is a variety of evidence that histone acetylation and deacetylation are mechanisms by which transcriptional regulation is achieved in cells (Grunstein, M. (1997) Nature 389: 349-52). It is thought that by altering the affinity of histones for helical DNA in nucleosomes; these effects occur through changes in chromatin structure. 5 histones (designated H1, H2A, H2B, H3 and H4) have been identified. Histones H2A, H2B, H3 and H4 are found in nucleosomes, and H1 is a linker located between the nucleosomes. Each nucleosome contains two histones in its core, except H1, which is present alone in the outer portion of the nucleosome structure. It is believed that binding affinity of histones to the phosphodna backbone is greater when histone acetylation is low. This affinity causes the DNA to bind tightly to histones, making it difficult for the DNA to transcribe regulatory elements and machinery. The regulation of the acetylation state occurs through an active balance between two enzyme complexes Histone Acetyltransferase (HAT) and Histone Deacetylase (HDAC). It is thought that the low acetylation state inhibits transcription of the relevant DNA. This hypoacetylation state is catalyzed by the macroprotein complex including HDAC enzymes. HDACs are particularly shown to catalyze the removal of acetyl groups from chromatin nuclear histones.

It is believed that SAHA inhibition of HDAC occurs through direct interaction with the catalytic site of the enzyme as demonstrated by X-ray crystallography (Finnin, M.S., Donigian, J.R., Cohen, A. et al (1999) Nature 401: 188-. It is believed that the results of inhibiting HDAC have no general effect on the genome, but only affect a small subset of the genome (Van Lint, C., Emiliani, S., Verdin, E. (1996) Gene Expression 5: 245-53). Evidence provided by DNA micro-sequencing (microarrays) of malignant c tumor cell lines cultured with HDAC inhibitors suggests a limited number (1-2%) of genes whose products are altered. For example, treatment of cells with HDAC inhibitors in culture appears to continuously induce the cyclin-dependent kinase inhibitor p21(Archer, S.Shufen, M.Shei, A., Hodin, R. (1998) PNAS 95: 6791-96). The protein plays an important role in inhibiting the cell cycle. It is believed that HDAC inhibitors increase the p21 transcription rate by spreading the highly acetylated state of histones in the p21 gene region, thus allowing the gene to transcribe machinery. Genes whose expression is not affected by HDAC inhibitors are not altered in acetylation of regioassociated histones (Dressel, U., Renkawitz, R., Baniahmad, A. (2000) Anticancer Research 20 (2A): 1017-22).

In several instances, it has been shown that disruption of HAT or HDAC activity is involved in the development of a malignant phenotype. For example, in acute promyelocytic leukemia, the tumor protein resulting from fusion of PML and RAR α appears to inhibit specific gene transcription by recruiting HDACs (Lin, R.J., Nagy, L., Inoue, S. et al (1998) Nature 391: 811-14). In this way, the tumor cells cannot complete differentiation, resulting in hyperproliferation of the leukemia cell line.

U.S. Pat. nos. 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 tumor cells, which compounds have two polar end groups separated by a flexible chain of methylene groups or by a rigid phenyl group, wherein one or both of the polar end groups are large hydrophobic groups. In enzymatic assays, certain compounds also have the same large hydrophobic group at the same end of the molecule as the first hydrophobic group, which further increases differentiation activity by about 100-fold, and in cell differentiation assays by about 50-fold. Methods for synthesizing compounds for use in the methods and pharmaceutical compositions of the present invention are fully described in the aforementioned patents, the entire contents of which are incorporated herein by reference.

Current tumor therapy is known to consist of combined treatment of patients with more than one anti-tumor therapeutic agent. Examples are the use of radiotherapy in combination with chemotherapy and/or cytotoxic agents, more recently immunotherapy, for example with tumour cell specific therapeutic antibodies, in combination with radiotherapy. However, the possibility of combining treatments with each other to identify such combinations as being more effective than either method alone requires extensive preclinical and clinical trials, and without such trials it is impossible to predict which combinations show additive or even synergistic effects.

In addition to the purpose of increasing therapeutic efficacy, another purpose of combination therapy is that it is possible to reduce the dosage of each component in the resulting combination in order to reduce unwanted or harmful side effects caused by large doses of each component.

There is an urgent need to find suitable methods for treating cancer, including combination therapies that result in reduced side effects and effective treatment and control of malignancies.

Summary of The Invention

The present invention is based on the discovery that Histone Deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), can be used in combination with one or more anticancer agents to provide therapeutically effective anticancer effects.

It has been unexpectedly found that a combination of a first treatment regimen (procedure) comprising the administration of an HDAC inhibitor as described herein and a second treatment regimen with one or more anticancer agents as described herein provides a therapeutically effective anticancer effect. Each treatment (administration of the HDAC inhibitor and administration of the anti-cancer agent) is used in an amount or dose that provides a therapeutically effective treatment in combination with the other treatments.

Combination therapy may act by inducing differentiation, cell growth arrest and/or apoptosis of cancer cells. In addition, the effect of the HDAC inhibitor and the anti-cancer agent may be additive or synergistic. Combination therapy is particularly advantageous because the dose of each drug can be reduced in combination therapy compared to single drug therapy, while still achieving an overall anti-tumor effect.

Accordingly, 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 suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof in a first treatment regimen, and a second amount of an anti-cancer agent in a second treatment regimen, wherein the first and second amounts together comprise a therapeutically effective amount.

Treating cancer as used herein refers to partially or completely inhibiting, delaying or preventing the progression of cancer including metastasis in a mammal such as a human; inhibiting, delaying or preventing cancer recurrence including metastasis; or to prevent the onset or development of cancer (chemoprevention).

The methods of the invention are useful for treating a variety of cancers, including but not limited to, solid tumors (e.g., lung, breast, colon, prostate, bladder, rectal, brain, or endometrial tumors), hematologic malignancies (e.g., leukemias, lymphomas, myelomas), carcinomas (e.g., bladder, renal, breast, colorectal), neuroblastoma, or melanoma. Non-limiting examples of such cancers include cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphomas associated with human T-lymphotropic virus (HTLV), adult T-cell leukemia/lymphoma (ATLL), acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic myelogenous leukemia, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, mesothelioma; solid tumors of childhood such as brain neuroblastoma, retinoblastoma, Wilms' tumor, bone cancer, and soft tissue sarcoma; adult common solid tumors such as head and neck cancers (e.g., mouth, throat, and esophageal cancers), genitourinary cancers (e.g., prostate, bladder, kidney, uterus, ovary, testis, rectum, and colon cancers), lung cancers, breast cancers, pancreatic cancers, melanoma, and other skin cancers; gastric, brain, liver, adrenal, kidney, thyroid, basal cell, ulcerative and papillary squamous cell carcinoma, metastatic skin carcinoma, medullary carcinoma, osteosarcoma, ewings 'sarcoma, veticus cell sarcoma, kaposi's sarcoma, neuroblastoma and retinoblastoma.

The method comprises administering to a subject in need thereof a first amount of an HDAC inhibitor, e.g., SAHA, using a first treatment regimen and a second amount of an anti-cancer agent using a second treatment regimen. The first and second treatments together comprise a therapeutically effective amount.

The invention also relates to a pharmaceutical combination for the treatment of cancer. The pharmaceutical combination includes a first amount of an HDAC inhibitor, e.g., SAHA, and a second amount of an anti-cancer agent. The first and second amounts together comprise a therapeutically effective amount.

The invention also relates to the use of a first amount of an HDAC inhibitor and a second amount of an anti-cancer agent in the manufacture of a medicament for the treatment of cancer.

In a particular embodiment of the invention, the combination of the HDAC inhibitor and the anti-cancer agent is additive, i.e., the combination treatment regimen produces an additive effect as a result of the individual components being administered separately. According to this embodiment, the amount of the HDAC inhibitor and the amount of the anti-cancer agent together comprise an effective amount to treat cancer.

In another embodiment of the invention, the combination of the HDAC inhibitor and the anti-cancer agent is considered therapeutically synergistic when the combination regimen produces significantly better anti-cancer results (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death) than the additive effects of the individual components when administered alone at therapeutic doses. When the results are significantly better, they can be determined by standard statistical analysis. For example, the Mann-Whitney test or some other generally accepted statistical analysis may be employed.

The treatment regimen may be performed sequentially in any order, simultaneously, or a combination thereof. For example, a first treatment regimen of administering the HDAC inhibitor can be performed before a second treatment regimen, i.e., the anti-cancer agent, after a second treatment regimen of the anti-cancer agent, concurrently with a second treatment regimen of the anti-cancer agent, or a combination thereof. For example, the total course of therapy of the HDAC inhibitor can be determined. The anti-cancer agent may be administered before the start of treatment with the HDAC inhibitor or after treatment with the HDAC inhibitor. In addition, the anti-cancer treatment may be administered during the administration of the HDAC inhibitor, but need not occur during the entire HDAC inhibitor treatment. Likewise, the HDAC inhibitor treatment can be administered during the administration of the anti-cancer agent, but need not occur during the entire anti-cancer agent treatment period. In another embodiment, the treatment regimen comprises a pretreatment with one drug of an HDAC inhibitor or an anti-cancer drug followed by the addition of a second drug during the treatment period.

In a particular embodiment of the invention, the HDAC inhibitor may be administered in combination with any one or more other HDAC inhibitors, alkylating agents, antibiotics, antimetabolites, hormonal agents, plant derived agents, anti-vascular agents, differentiation inducers, cell growth arrest inducers, apoptosis inducers, cytotoxic agents, biological agents, gene therapy agents, or any combination thereof.

In a particular embodiment of the invention, the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA), which can be administered in combination with any one or more additional HDAC inhibitors, alkylating agents, antibiotics, antimetabolites, hormonal agents, plant-derived agents, anti-angiogenic agents, differentiation inducers, cell growth arrest inducers, apoptosis inducers, cytotoxic agents, biological agents, gene therapy agents, or any combination thereof.

HDAC inhibitors suitable for use in the present invention include, but are not limited to, hydroxamic acid derivatives, Short Chain Fatty Acids (SCFAs), cyclic tetrapeptides, benzamide derivatives, or electrophilic ketone derivatives as defined herein. Specific non-limiting examples of HDAC inhibitors suitable for use in the methods of the present invention are:

A) a hydroxamic acid derivative selected from the group consisting of: SAHA, Pyroxamide, CBHA, trichostatin A (TSA), trichostatin C, salicyloylbis-Hydroxamic Acid, Azelaic Acid (Azelaic Bishydroxamic Acid) (ABHA), Azelaic Acid-1-Hydroxamate-9-Anilide (Azelaic-1-Hydroxamate-9-Anilide) (AAHA), 6- (3-chlorophenylureido) hexanoic Acid (carbomic Acid) (3Cl-UCHA), Oxamfllatin, A-161906, Scriptaid, PXD-101, LAQ-824, CHAP, MW2796 and MW 2996;

B) a cyclic tetrapeptide selected from: trapoxin a, FR901228(FK228 or depsipeptide), FR225497, Apicidin, CHAP, HC-toxin, WF27082, and Chlamydocin;

C) short Chain Fatty Acids (SCFAs) selected from: sodium butyrate, isovalerate, valerate, 4-phenylbutyrate (4-PBA), Phenylbutyrate (PB), propionate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, tributyrin, valproic acid, and valproate;

D) a benzamide derivative selected from: CI-994, MS-27-275(MS-275) and 3' -amino derivatives of MS-27-275;

E) an electrophilic ketone derivative selected from the group consisting of: trifluoromethyl ketones and α -ketoamides such as N-methyl- α -ketoamide; and

F) including natural products, psammaplins and Depudecin.

Specific HDAC inhibitors include:

suberoylanilide hydroxamic acid (SAHA) represented by the following structural formula:

pyroxamide represented by the following structural formula:

m-carboxycinnamic acid dihydroxyamide (CBHA) represented by the following structural formula:

other non-limiting examples of HDAC inhibitors suitable for use in the methods of the present invention are:

a compound represented by the following structure:

wherein R is₃And R₄Independently substituted or unsubstituted: branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkoxy, aryloxy, arylalkoxy or pyridyl; cycloalkyl, aryl, aryloxy, arylalkoxy or pyridyl, or R₃And R₄Taken together to form a piperidinyl group; r₂Is a hydroxyamino group; and n is an integer from 5 to 8.

A compound represented by the following structure:

wherein R is the following substituted or unsubstituted group: phenyl, piperidine, thiazole, 2-pyridine, 3-pyridine or 4-pyridine, and n is an integer of 4 to 8.

A compound represented by the following structure:

wherein A is an amide moiety, R₁And R₂Each selected from substituted or unsubstituted aryl, arylalkyl, naphthyl, pyridylamino, 9-purin-6-amino, thiazolylamino, aryloxy, arylalkoxy, pyridyl, quinolinyl or isoquinolinyl; r₄Is hydrogen, halogen, phenyl or cycloalkyl moiety and n is an integer from 3 to 10.

Alkylating agents suitable for use in the present invention include, but are not limited to, dichloroethanes (nitrogen mustards such as chlorambucil, cyclophosphamide, ifosfamide, nitrogen mustards, melphalan, uramustine), aziridines (e.g., thiotepa), alkyl ketosulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, streptozocin), non-classical alkylating agents (altretamine, dacarbazine and procarbazine), platinum compounds (carboplatin and cisplatin).

Antibiotics suitable for use in the present invention are anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, and anthracenedione), mitomycin C, bleomycin, actinomycin D, plicatomycin.

Antimetabolites suitable for use in the present invention include, but are not limited to, floxuridine, fluorouracil, methotrexate, leucovorin, hydroxyurea, thioguanine, mercaptopurine, cytarabine, pentostatin, fludarabine phosphate, cladribine, asparaginase, and gemcitabine. In a particular embodiment, the antimetabolite is gemcitabine.

Hormonal agents suitable for use in the present invention include, but are not limited to, estrogens, progestogens, antiestrogens, androgens, antiandrogens, LHRH analogs, aromatase inhibitors, diethylstilbestrol, tamoxifen, toremifene, fluoxymesterol, raloxifene, bicalutamide, nilutamide, flutamide, aminoglutethimide, tetrazoles, ketoconazole, goserelin acetate, leuprolide, megestrol acetate, and mifepristone.

Botanical drugs suitable for use in the present invention include, but are not limited to, vincristine, vinblastine, vindesine, vinorelbine, etoposide, teniposide, paclitaxel and docetaxel.

Biological agents suitable for use in the present invention include, but are not limited to, immunomodulatory proteins, monoclonal antibodies against tumor antigens, tumor suppressor genes, and cancer vaccines. For example, the immunomodulatory protein can be interleukin 2, interleukin 4, interleukin 12, interferon E1, interferon D, interferon alpha, erythropoietin, granulocyte-CSF; granulocytes, macrophage-CSF; bacillus Cahnette-Guerin, levamisole or octreotide. In addition, the tumor suppressor gene may be DPC-4, NF-1, NF-2, RB, p53, WT1, BRCA, or BRCA 2.

The HDAC inhibitor (e.g., SAHA) and the anti-cancer agent may be administered by any method of administration known to those skilled in the art. Examples of routes of administration include, but are not limited to, oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, liposomal; by inhalation, vaginal, intraocular; local delivery through a catheter or impression (stent), subcutaneous, intralipid, intrathecal, or sustained release dosage forms.

Of course, the route of administration of any of the SAHA or other HDAC inhibitors is independent of the route of administration of the anti-cancer agent. The presently preferred route of administration of SAHA is oral. Thus, according to this embodiment, SAHA is administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, buccally, intranasally, via liposomes; by inhalation, vaginal, intraocular; the second drug (anticancer drug) is administered via catheter or impression for local release, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form.

SAHA or any of the HDAC inhibitors can be administered at any dose and dosing regimen, along with the effects of the anti-cancer agent, to achieve a dose effective to treat cancer. For example, the total daily dose of SAHA or any HDAC inhibitor administered may be up to 800mg, preferably orally, once, twice and three times daily, continuously (daily) or intermittently (e.g. 3-5 days/week).

Accordingly, the present invention relates to a method of treating cancer in a patient in need thereof by administering to a patient in need thereof a first amount of suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof in a first treatment regimen in a total daily dose of up to 800mg, and a second amount of an anticancer agent in a second treatment regimen, wherein the first and second amounts together comprise a therapeutically effective amount.

In one embodiment, the HDAC inhibitor, e.g., SAHA, is administered in a pharmaceutical composition, preferably suitable for oral administration. In a presently preferred embodiment, SAHA is administered orally in a gelatin capsule, which may contain excipients such as microcrystalline cellulose, croscarmellose sodium, and magnesium stearate.

The HDAC inhibitor can be administered in a total daily dose, which can vary from patient to patient, and in different dosage regimens. Suitable dosages for oral administration are about 25-4000mg/m total daily dose²Once, twice or three times daily, continuously (daily) or intermittently (e.g. 3-5 days/week). In addition, the compositions can be administered on a periodic basis with drug withdrawal periods between periods (e.g., 2-8 weeks of treatment with up to 1 week between treatments).

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

Any one or more specific doses and dosage regimens of the HDAC inhibitor will be apparent to those skilled in the art and are applicable to any one or more anti-cancer agents used in combination therapy. In addition, the specific dose and dosage regimen of the anti-cancer agent may also be varied, and the optimal dose, dosage regimen and route of administration will depend on the particular anti-cancer agent used.

The present invention also provides a method of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells and 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 regimen, and a second amount of an anti-cancer agent in a second treatment regimen, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest, apoptosis of the cells.

The present invention also provides an in vitro method of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells to inhibit proliferation of such cells by contacting the cells with a first amount of suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof and a second amount of an anticancer agent, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest, apoptosis of the cells.

In view of the differential toxicity associated with the two treatment modalities, combination therapy may provide therapeutic advantages. For example, treatment with HDAC inhibitors may result in exceptional toxicity not present in anticancer drugs, and vice versa. Thus, this differential toxicity may allow each treatment to be administered at a dose where the toxicity is absent or minimal, such that the combination therapies together provide the therapeutic dose while avoiding toxicity of the individual components of the combination. Moreover, when a therapeutic effect is achieved, e.g., significantly better than additive, due to the potentiation or synergy of the combination therapy, the dosage of each agent can even be reduced, thereby reducing the associated toxicity to a greater extent.

Brief Description of Drawings

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different angles. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1: the role of SAHA and gemcitabine combination in the T24 cell line. As described in the experimental part, at the indicated time points, the cells were untreated (□), treated with 2nM gemcitabine (. diamond.), treated with 5. mu.M SAHA (. smallcircle.), or treated with a combination of 2nM gemcitabine and 5. mu.M SAHA (. DELTA.). FIG. 1A shows cell proliferation and FIG. 1B shows cell viability.

FIG. 2: effect of SAHA and gemcitabine combination in LnCaP cell line. As described in the experimental section, at the indicated time points, (□) indicates untreated cells, (. diamond) indicates treatment with 2nM gemcitabine, (. o) indicates treatment with 5. mu.M SAHA, or (. DELTA.) indicates treatment with a combination of 2nM gemcitabine and 5. mu.M SAHA. FIG. 2A shows cell proliferation and FIG. 2B shows cell viability.

FIG. 3: the role of SAHA and 5-azacytidine in combination in the T24 cell line. As described in the experimental part, at the indicated time points, (□) indicates untreated cells, (. diamond) indicates treatment with 200nM 5-Azacytidine (AZ), (. smallcircle) indicates treatment with 5. mu.M SAHA, or (. DELTA.) indicates treatment with a combination of 200nM 5-azacytidine and 5. mu.M SAHA. FIG. 3A shows cell proliferation and FIG. 3B shows cell viability. Asterisks indicate time points of addition of SAHA.

FIG. 4: effect of SAHA combination on MDA-231 cell proliferation. FIG. 4A: cells were pretreated with SAHA at the indicated concentration for 4 hours, washed, and then the second drug was added for 48 hours. FIG. 4B: cells were pretreated with SAHA at the indicated concentration for 48 hours, the second drug was added for 4 hours, and then cells were washed. After 48 hours, cell growth was quantified using MTS.

FIG. 5: effect of SAHA combination on proliferation of DU145 cells. Cells were pretreated with SAHA at the indicated concentration for 48 hours, the second drug was added for 4 hours, and then cells were washed. After 48 hours, cell growth was quantified using MTS.

FIG. 6: effect of SAHA combination on the clonal development of DU145 cells. Cells were treated with SAHA for 48 hours, then the second drug was added for 4 hours, and then the cells were washed. Colony formation was assessed after 2-3 weeks.

FIG. 7: effect of SAHA combination on MDA-231 cell clonogeny. Cells were treated with SAHA for 48 hours, the second drug was added for 4 hours, and then cells were washed. Colony formation was assessed after 2-3 weeks.

FIG. 8: effect of SAHA combination on U118 cell clonogeny. Cells were treated with SAHA for 48 hours, the second drug was added for 4 hours, and then cells were washed. Colony formation was assessed after 2-3 weeks.

FIG. 9: percent inhibition of LnCap cells treated with SAHA and irinotecan. Cells were incubated with SAHA alone at the indicated concentrations; incubation with irinotecan alone; and incubation in combination with SAHA and irinotecan. The histogram on the right hand side of each experiment represents the calculated effect of the additive interaction.

FIG. 10: percentage inhibition of LnCap cells treated with SAHA and 5-Fluorouracil (5-FU). Cells were incubated with SAHA alone at the indicated concentrations; incubation with 5-FU alone; and incubating in combination with SAHA and 5-FU. The histogram on the right hand side of each experiment represents the calculated effect of the additive interaction.

FIG. 11: percentage inhibition of LnCap cells treated with SAHA and docetaxel. Cells were incubated with SAHA alone at the indicated concentrations; incubation with docetaxel alone; and incubation in combination with SAHA and docetaxel. The histogram on the right hand side of each experiment represents the calculated effect of the additive interaction.

Detailed Description

The present invention relates to a method of treating cancer in a subject in need thereof by administering to the subject in need thereof a first amount of an HDAC inhibitor or a pharmaceutically acceptable salt or hydrate thereof in a first treatment regimen and a second amount of an anti-cancer agent in a second treatment regimen, wherein the first and second amounts together comprise a therapeutically effective amount. The effects of the HDAC inhibitor and the anti-cancer agent may be additive or synergistic.

The present invention also relates to a method of treating cancer in a subject in need thereof by administering to the subject in need thereof a first amount of suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof in a first treatment regimen and a second amount of an anti-cancer agent in a second treatment regimen, wherein the first and second amounts together comprise a therapeutically effective amount. The effects of SAHA and anticancer drugs may be additive or synergistic.

The various grammatical forms of the term "treatment" in connection with the present invention refer to preventing (i.e., chemopreventing), curing, reversing, alleviating, minimizing, inhibiting or stopping the deleterious effects of a disease state, disease progression, disease pathogen (e.g., bacteria or virus), or other abnormal condition. For example, treatment may involve alleviating symptoms of the disease (i.e., not necessarily all symptoms) or slowing disease progression. Because certain of the methods of the present invention involve physical removal of the pathogen, the skilled artisan will recognize that they are equally effective in situations where the compounds of the present invention are administered prior to or simultaneously with exposure to the pathogen (prophylactic treatment) and after exposure to the pathogen (even after rehabilitation).

Cancer treatment as used herein refers to the partial or complete inhibition, delay or prevention of the progression of cancer, including metastasis; inhibiting, delaying or preventing recurrence of cancer, including cancer metastasis; or to prevent (chemoprevent) the onset or development of cancer in a mammal, such as a human. In addition, the methods of the invention will be useful for the chemopreventive treatment of human cancer patients. However, the methods of the invention may also be effective in treating cancer in other mammals.

The term "therapeutically effective amount" as used herein refers to the combined amount of the first and second treatments in a combination therapy that is effective. The combined amounts achieve the desired biological response. In the present invention, the desired biological response is a partial or complete inhibition, delay or prevention of the progression of cancer, including metastasis; inhibiting, delaying or preventing the recurrence of cancer, including metastasis; or to prevent (chemoprevent) the onset or development of cancer in a mammal, such as a human.

The terms "combination therapy", "combination therapy" and "treatment" are used interchangeably herein to refer to the treatment of an individual with at least two different therapeutic agents. According to the invention, the subject is treated with a first therapeutic agent, preferably SAHA or another HDAC inhibitor described herein. The second therapeutic agent may be another HDAC inhibitor, or may be any clinically established anti-cancer agent as defined herein. The combination therapy may include a third or even more therapeutic agents.

The method comprises administering to a patient in need thereof a first amount of a histone deacetylase inhibitor, such as SAHA, in a first treatment regimen, and administering a second amount of an anti-cancer agent in a second treatment regimen. The first and second treatments together comprise a therapeutically effective amount.

The invention also relates to a pharmaceutical combination for the treatment of cancer. The pharmaceutical combination comprises a first amount of an HDAC inhibitor, such as SAHA, and a second amount of an anti-cancer agent. The first and second amounts together comprise a therapeutically effective amount.

In a particular embodiment of the invention, the HDAC inhibitor and anticancer agent are combined additively, i.e. the combination treatment regimen produces an additive effect as a result of the separate administration of the individual components. According to this embodiment, the amount of the HDAC inhibitor and the amount of the anti-cancer agent together comprise an effective amount to treat cancer.

In another embodiment of the invention, the combination of the HDAC inhibitor and the anticancer agent is considered therapeutically synergistic when the combination therapy regimen produces significantly better anticancer results (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death) than the additive effects of each component when administered alone at therapeutic doses. When the results are significantly better, they can be determined by standard statistical analysis. For example, the Mann-Whitney test or some other generally accepted statistical analysis may be used.

The treatment regimen may be performed sequentially in any order, simultaneously, or a combination thereof. For example, the HDAC inhibitor of the first treatment regimen can be administered prior to the second treatment regimen, i.e., the anti-cancer agent, after the second treatment with the anti-cancer agent, concurrently with the second treatment with the anti-cancer agent, or a combination thereof. For example, the total course of therapy for the HDAC inhibitor can be determined. The anti-cancer agent may be administered before the start of treatment with the HDAC inhibitor or after treatment with the HDAC inhibitor. In addition, the anti-cancer treatment may be administered during the administration of the HDAC inhibitor, but need not occur during the entire HDAC inhibitor treatment. In another embodiment, the treatment regimen comprises a pretreatment with one drug of an HDAC inhibitor or an anti-cancer drug followed by the addition of a second drug.

The methods of the invention are useful for treating a variety of cancers, including but not limited to solid tumors (e.g., tumors of the lung, breast, colon, prostate, bladder, rectum, brain, or endometrium); hematological malignancies (e.g., leukemia, lymphoma, myeloma), cancers (e.g., bladder cancer, renal cancer, breast cancer, colorectal cancer), neuroblastoma, or melanoma. Non-limiting examples of such cancers include cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphomas associated with human T-lymphotropic virus (HTLV), adult T-cell leukemia/lymphoma (ATLL), acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic myelogenous leukemia, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, mesothelioma; solid tumors of childhood such as brain neuroblastoma, retinoblastoma, Wilms' tumor, bone cancer, and soft tissue sarcoma; adult common solid tumors such as head and neck cancers (e.g., mouth, throat, and esophagus), genitourinary cancers (e.g., prostate, bladder, kidney, uterus, ovary, testis, rectum, and colon cancers), lung cancers, breast cancers, pancreatic cancers, melanoma, and other skin cancers; gastric, brain, liver, adrenal, kidney, thyroid, basal cell, ulcerative and papillary squamous cell carcinoma, metastatic skin carcinoma, medullary carcinoma, osteosarcoma, ewings 'sarcoma, veticus cell sarcoma, kaposi's sarcoma, neuroblastoma and retinoblastoma.

In a particular embodiment of the invention, the HDAC inhibitor may be administered in combination with an additional HDAC inhibitor. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with an alkylating agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with an antibiotic agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with an antimetabolite. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with a hormonal agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with a plant-derived drug. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with an antiangiogenic agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with a differentiation-inducing agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with an inducer of cell growth arrest. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with an apoptosis inducing agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with a cytotoxic agent. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with a biologic. In another embodiment of the invention, the HDAC inhibitor may be administered in combination with any combination of an additional HDAC inhibitor, an alkylating agent, an antibiotic, an antimetabolite, an 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, or a biologic.

In a particular embodiment of the invention, the HDAC inhibitor is SAHA, which may be administered in combination with any one or more other HDAC inhibitors, alkylating agents, antibiotics, antimetabolites, hormonal agents, plant-derived agents, anti-angiogenic agents, differentiation inducing agents, cell growth arrest inducing agents, apoptosis inducing agents, cytotoxic agents, biological agents, gene therapy agents, or any combination thereof.

In view of the differential toxicity associated with the two treatment modalities, combination therapy may provide therapeutic advantages. For example, treatment with HDAC inhibitors may result in exceptional toxicity not present with anticancer drugs and vice versa. Thus, this differential toxicity may allow each treatment to be administered at a dose where the toxicity is absent or minimal, such that the combination therapies together provide the therapeutic dose while avoiding toxicity of the individual components of the combination. Moreover, when a therapeutic effect is obtained as a result of an enhancement or synergy of the combination therapy, e.g., significantly better than additive, the dosage of each drug can be even further reduced, thus reducing the associated toxicity to a greater extent.

Histone deacetylase and histone deacetylase inhibitors

The term Histone Deacetylases (HDACs) as used herein is an enzyme that catalyzes the removal of the acetyl group in a lysine residue at the amino terminus of the nucleosome histone. Thus, HDACs together with Histone Acetyltransferases (HATs) regulate the acetylation status of histones. Histone acetylation affects gene expression, and HDACs inhibitors such as the hydroxamic acid-based hybrid polar compound suberoylanilide hydroxamic acid (SAHA) induce growth arrest, differentiation and/or apoptosis in transformed cells in vitro, inhibiting tumor growth in vivo. HDACs can be classified into three groups according to structural homology. Class I HDACs (HDACs 1, 2, 3 and 8) share yeast RPD3 protein similarity, 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 yeast HDA1 protein and have nuclear and cytoplasmic subcellular localization. Hydroxamic acid-based HDAC inhibitors such as SAHA inhibit class I and II HDACs. Class III HDACs form structurally dissimilar NAD-dependent enzymes that are associated with the yeast SIR2 protein and are not inhibited by hydroxamic acid-based HDAC inhibitors.

The term histone deacetylase inhibitor or HDAC inhibitor as used herein is a compound capable of inhibiting histone deacetylation in vivo, in vitro or both. Thus, HDAC inhibitors inhibit the activity of at least one histone deacetylase. As a result of inhibiting deacetylation of at least one histone, an increase in acetylated histone occurs and accumulation of acetylated histone is a suitable biomarker for assessing HDAC inhibitor activity. Therefore, a method for measuring the accumulation of acetylated histones can be used to measure the HDAC inhibitory activity of a compound of interest. It will be appreciated that compounds which inhibit histone deacetylase activity may also bind to other substrates and may therefore inhibit other biologically active molecules such as enzymes. It is also understood that the compounds of the invention are capable of inhibiting any of the histone deacetylases set forth above or any other histone deacetylase.

For example, in patients receiving HDAC inhibitors, acetylated histone accumulation in peripheral monocytes and tissues treated with HDAC inhibitors can be measured using appropriate controls.

The HDAC inhibitory activity of a particular compound can be determined in vitro, for example, using an enzymatic assay that exhibits inhibition of at least one histone deacetylase. In addition, measurement of the accumulation of acetylated histones in cells treated with the subject compositions can measure the HDAC inhibitory activity of the compounds.

Methods for measuring the accumulation of acetylated histones are well known in the literature. See, e.g., Marks, p.a., et al, j.natl.cancer inst.92: 1210-1215, 2000, Butler, L.M., et al, Cancer Res.60: 5165-; richon, v.m., et al, proc.natl.acad.sci., USA, 95: 3003-3007, 1998 and Yoshida, m, et al, j.biol.chem., 265: 17174-17179, 1990.

For example, an enzymatic assay for determining the activity of an HDAC inhibitor compound can be performed as follows. Briefly, the effect of HDAC inhibitor compounds on the affinity of purified human epitopic (flag) HDAC1 can be determined by incubating enzyme preparations with a defined amount of inhibitor compounds for about 20 minutes on ice in the absence of substrate. Can be added with the substrate ([ 2 ]³H]Acetyl-labeled histone derived from murine erythroleukemia cells), the sample can be incubated at 37 ℃ for 20 minutes in a total volume of 30 μ L. The reaction can then be terminated and the acetate released can be extracted and the amount of radiation released can be determined by scintillation counting. Another assay for determining the activity of HDAC inhibitor compounds is from"HDAC fluorescence Activity assay of Research Laboratories, Inc., PlymouthMeeting, Pa; drug discovery kit (Drug discovery kit) -AK-500'.

In vivo studies can be performed as follows. Animals such as mice can be injected intraperitoneally with an HDAC inhibitor compound. Following administration, selected tissues such as brain, spleen, liver, etc. may be isolated at predetermined times. Can be prepared from a mixture essentially similar to Yoshida et al, j.biol.chem.265: 17174 histones were isolated from tissues described in 17179, 1990. Equivalent amounts of histones (about 1. mu.g) can be electrophoresed on a 15% SDS-polyacrylamide gel and then transferred to a Hybond-P filter (from Amersham). The filters can be blocked with 3% milk and probed with rabbit purified polyclonal anti-acetylated histone H4 antibody (α -Ac-H4) and anti-acetylated histone H3 antibody (α -Ac-H3) (Upstate Biotechnology, Inc.). Acetylated histone levels can be visually examined with goat anti-rabbit antibody conjugated to horseradish peroxidase (1: 5000) and SuperSignal chemiluminescent substrate (Pierce). As a loading control for histones, parallel gel electrophoresis followed by staining with Coomassie Blue (CB) can be performed.

In addition, hydroxamic acid based HDAC inhibitors have been shown to upregulate p21^WAF1And (4) expressing the gene. A variety of transformed cells were incubated with HDAC inhibitors using standard procedures and induced within 2 hoursp21^WAF1A protein. p21^WAF1Gene induction is associated with the accumulation of acetylated histones in the chromatin region of the gene. Thus, p21 can be considered^WAF1Induction involved G1 cell cycle arrest in transformed cells caused by HDAC inhibitors.

HDAC inhibitors are effective in treating a wide range of diseases characterized by neoplastic cell proliferation, such as any of the cancers described above. However, the therapeutic use of HDAC inhibitors is not limited to the treatment of cancer. HDAC inhibitors have been found to be useful in a wide variety of diseases.

For example, HDAC inhibitors, and in particular SAHA, have been found to be useful in the treatment of various acute and chronic inflammatory diseases, autoimmune diseases, allergic diseases, diseases associated with oxidative stress, and diseases characterized by cellular hyperproliferation. Non-limiting examples of joint inflammation include Rheumatoid Arthritis (RA) and psoriatic arthritis; inflammatory bowel diseases such as crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis (including T cell mediated psoriasis) and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous, and allergic vasculitis); eosinophilic myositis, eosinophilic fasciitis; cancer with leukocyte infiltration of the skin or organ, ischemic injury including cerebral ischemia (e.g., trauma-induced brain injury, epilepsy, hemorrhage, or stroke, each of which can lead to neurodegeneration); HIV, heart failure; chronic, acute or malignant liver disease, autoimmune thyroiditis; systemic lupus erythematosus, Sjorgren's syndrome, pulmonary diseases (e.g., ARDS); acute pancreatitis; amyotrophic Lateral Sclerosis (ALS); alzheimer's disease; cachexia/anorexia; asthma; atherosclerosis; chronic fatigue syndrome, fever; diabetes (e.g., insulin diabetes or juvenile diabetes); glomerulonephritis; graft-versus-host rejection (e.g., in transplant surgery); hemorrhagic shock; hyperalgesia; inflammatory bowel disease; multiple sclerosis; myopathies (e.g. muscle protein metabolism, especially in sepsis); osteoporosis; parkinson's disease; pain; premature delivery; psoriasis; reperfusion injury; cytokine-induced toxicity (e.g., septic shock, endotoxic shock); side effects caused by radiotherapy, temporal mandibular joint disease, tumor metastasis; or inflammation resulting from strain, sprain, cartilage damage, trauma such as burns, orthopedic surgery, infection, or other disease processes. Allergic diseases and disorders include, but are not limited to, respiratory allergic diseases such as asthma, allergic rhinitis, allergic lung disease, allergic pneumonia, eosinophilic pneumonia (e.g., Loeffler's syndrome, chronic eosinophilic pneumonia), delayed-type hypersensitivity, Interstitial Lung Disease (ILD) (e.g., idiopathic pulmonary fibrosis or ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis, or dermatomyositis); systemic anaphylaxis or hypersensitivity, drug allergies (e.g., to penicillins, cephalosporins), insect bite allergies, and the like.

For example, HDAC inhibitors, and particularly SAHA, have been found to be useful in the treatment of various neurodegenerative diseases, a non-exhaustive list of which is as follows:

I. disorders characterized by progressive dementia without other significant neurological signs, such as alzheimer's disease; senile dementia of the alzheimer type and Pick's disease (atrophy of the brain lobes).

Syndromes that combine progressive dementia with other apparent neurological abnormalities, such as: A) syndromes that occur predominantly in adults (e.g. huntington's disease, combined dementia and dyskinesia and/or parkinson's disease, progressive supranuclear palsy (Steel-Richardson-Olszewski), diffuse lewy body disease and multiple system atrophy of degenerative manifestations of the corticodentational nucleus); and B) syndromes that occur mainly in children or young adults (e.g. halloysit-Spatz (Hallervordeh-Spatz) disease and progressive familial myoclonic epilepsy).

Syndromes of progressive abnormalities of body state and movement, such as paralysis agitans (Parkinson's disease), striatonigral degeneration, progressive supranuclear palsy, torsion dystonia (torsion spasm; dystonia deformans), spastic torticollis and other movement disorders, familial tremors and Tourette's syndrome.

Syndromes of progressive movement disorders, such as cerebellar degeneration (e.g. cerebellar cortical degeneration and olivopontocerebellar atrophy (OPCA)); and spinocerebellar degeneration (Friedreich's) ataxia and related disorders).

Central autonomic nervous system failure syndrome (summer-dererger (Shy-Drager) syndrome).

Syndromes of muscle weakness and wasting without sensory changes (motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy (e.g. infantile spinal muscular atrophy (Werdnig-Hoffman), juvenile spinal muscular atrophy (Wohlfart-Kugelberg-Welander) and other forms of familial spinal muscular atrophy), primary lateral sclerosis and hereditary spastic paraplegia.

Syndromes combining sensory alterations of muscle weakness and wasting (progressive muscular atrophy; chronic familial polyneuropathy), such as peroneal muscular atrophy (Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy (Dejerine-Sottas) and various forms of chronic progressive neuropathy.

Progressive visual loss syndromes, such as retinitis pigmentosa (retinitis pigmentosa) and hereditary optic atrophy (Leber's) disease).

Generally, HDAC inhibitors are generally classified into 5 classes: 1) hydroxamic acid derivatives; 2) short Chain Fatty Acids (SCFAs); 3) a cyclic tetrapeptide; 4) benzamide and 5) electrophilic ketone.

Accordingly, the present invention includes within its broad scope compositions comprising an HDAC inhibitor which is 1) a hydroxamic acid derivative and/or any other compound capable of inhibiting histone deacetylase; 2) short Chain Fatty Acids (SCFAs); 3) a cyclic tetrapeptide; 4) a benzamide; 5) an electrophilic ketone; for inhibiting histone deacetylase, inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and/or inducing differentiation, cell growth arrest and/or apoptosis of neoplastic cells.

Non-limiting examples of such HDAC inhibitors are listed below. It is to be understood that the present invention encompasses any salt, crystalline structure, amorphous structure, hydrate, derivative, metabolite, stereoisomer, structural isomer, and prodrug of the HDAC inhibitor described herein.

A.Hydroxamic acid derivativesFor example suberoylanilide hydroxamic acid (SAHA) (Richon et al, Proc. Natl. Acad. Sci. USA 95, 3003-; m-carboxy cinnamic acid dihydroxyamide (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); salicyloylhydroxamic acids (Andrews et al, International J.Parasitology 30, 761-768 (2000)); suberoyl hydroxamic acid (SBHA) (U.S. patent No. 5,608,108); azelaic acid (ABHA) (Andrews et al, supra); azelaic acid-1-hydroxamate-9-anilide (AAHA) (Qiu et al, mol. biol. Cell11, 2069. sub. 2083 (2000)); 6- (3-chlorophenylureido) hexoxamic acid (3 Cl-UCHA); oxamflatin [ (2E) -5- [3- [ (phenylsulfonyl) aminophenyl]-pent-2-en-4-ynoyl hydroxamic acid (ynohydroxamic acid)](Kim et al Oncogene, 18: 24612470 (1999)); a-161906, script (Su et al 2000 Cancer Research, 60: 3137-; 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. Pat. nos. 5,369,108, 5,932,616, 5,700,811, 6,087,367, and 6,511,990.

B.Cyclic tetrapeptidesSuch as trapoxin A (TPX) -cyclotetrapeptide (cyclo- (L-phenylalanyl-D- (2-methyl) piperidinyl-L-2-amino-8-oxo-9, 10-epioxydecanoyl)) (Kijima et al, J biol. chem.268, 22429-; 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(2 months and 17 days 2000)); apicidin cyclotetrapeptide [ cyclo (N-O-methyl-L-tryptophanyl-L-isoleucyl-D- (2-methyl) piperidinyl-L-2-amino-8-oxodecanoyl)](Darkin-Rattray et al, Proc. Natl. Acad. Sci. USA 93, 1314313147 (199)6) ); apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, and apicidin IIb (P.Dulski et al, PCT application WO 97/11366); CHAP, HC-toxin cyclotetrapeptide (Bosch et al, Plant Cell 7, 1941-1950 (1995)); WF27082 cyclotetrapeptide (PCT application WO 98/48825); and chlamydocin (Bosch et al, supra).

C.Short Chain Fatty Acid (SCFA) derivativesFor example sodium butyrate (Coosens et al, J.biol.chem.254, 1716-1723 (1979)); isovalerate (McBain et al, biochem. Pharm. 53: 1357-1368 (1997)); valeric acid salts (esters) (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 tulsky, supra); phenyl acetate (Lea and tulsky, supra); 3-bromopropionate (Lea and tulssan, supra); tributyrin (Guan et al, Cancer Research, 60, 749-; valproic acid, valproate and Pivanex^TM。

D.Benzamide derivativesSuch as CI-994; MS-275[ N- (2-aminophenyl) -4- [ N- (pyridin-3-ylmethoxycarbonyl) aminomethyl]Benzamide derivatives](Saito et al, Proc. Natl. Acad. Sci. USA 96, 4592-4597 (1999)); and 3' -amino derivatives of MS-275 (Saito et al, supra).

E.Electrophilic ketone derivativesFor example, trifluoromethyl ketone (Frey et al, Bioorganic&Med, chem, lett, (2002), 12, 3443-; U.S.6,511,990) and α -ketoamides such as N-methyl- α -ketoamide.

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

Preferred hydroxamic acid based HDAC inhibitors are suberoylanilide hydroxamic acid (SAHA), m-carboxy cinnamic acid dihydroxyamide (CBHA), and pyroxamide. SAHA was shown to bind directly in the catalytic pocket of histone deacetylase. SAHA induces cell cycle arrest, differentiation and/or apoptosis of transformed cells in culture and inhibits rodent tumor growth. SAHA effectively induces these effects in solid tumors and blood cancers. SAHA was shown to effectively inhibit tumor growth in animals, and is non-toxic to animals. SAHA-induced inhibition of tumor growth is associated with accumulation of acetylated histones in the tumor. SAHA effectively inhibits the development and continued growth of mammary tumors in rats induced by carcinogens (N-methylnitrosourea). In a 130 day study, rats were given SAHA-containing feed. Thus, SAHA is a non-toxic, orally active antineoplastic agent whose mechanism of action involves inhibiting histone deacetylase activity.

Preferred HDAC inhibitors are those disclosed in U.S. patent nos. 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990, issued to certain of the present inventors, the entire contents of which are incorporated herein by reference, non-limiting examples of which are set forth below:

in one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 1, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₁And R₂May be the same or different; when R is₁And R₂And, at the same time, each is substituted or unsubstituted arylamino, cycloalkylamino, pyridylamino, piperidino, 9-purin-6-amine or thiazolylamino; when R is₁And R₂Not simultaneously, R₁＝R₃-N-R₄Wherein R is₃And R₄Each independently of the others, identical or different from each other and is hydrogen, hydroxy, substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, arylalkoxy, aryloxy, arylalkoxy or pyridyl, or R₃And R₄Taken together to form a piperidinyl radical, R₂Is hydroxylamino, hydroxy, amino, alkylamino, dialkylamino or alkoxyAnd n is an integer from about 4 to about 8.

In a particular embodiment of formula 1, R₁And R₂Same, is substituted or unsubstituted thiazolylamino; and n is an integer from about 4 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 2, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₃And R₄Each independently of the others, identical or different from each other and is hydrogen, hydroxy, substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, arylalkoxy, aryloxy, arylalkoxy or pyridyl, or R₃And R₄Taken together to form a piperidinyl radical, R₂Is hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino or alkoxy, and n is an integer from about 4 to about 8.

In one embodiment of formula 2, R₃And R₄Each independently of the others, identical or different from each other and is hydrogen, hydroxy, substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkoxy, aryloxy, arylalkoxy or pyridyl, or R₃And R₄Taken together to form a piperidinyl group; r₂Is hydroxylamino, hydroxyl, amino, alkylamino or alkoxy; n is an integer of 5 to 7; r₃-N-R₄And R₂Different.

In another embodiment of formula 2, n is 6. In yet another embodiment of formula 2, R₄Is a hydrogen atom, R₃Is a substituted or unsubstituted phenyl group, and n is 6. In yet another embodiment of formula 2, R₄Is a hydrogen atom, R₃Is a substituted phenyl group, and n is6 wherein the substituents of the phenyl group are selected from the group consisting of methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2, 3-difluoro, 2, 4-difluoro, 2, 5-difluoro, 3, 4-difluoro, 3, 5-difluoro, 2, 6-difluoro, 1, 2, 3-trifluoro, 2, 3, 6-trifluoro, 2, 4, 6-trifluoromethyl, 3,4, 5-trifluoromethyl, 2, 3,5, 6-tetrafluoro, 2, 3,4, 5, 6-pentafluoro, azido, hexyl, tert-butyl, phenyl, carboxyl, hydroxyl, methoxy, phenoxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methoxycarbonyl, methylaminocarbonyl, dimethylamino, dimethylaminocarbonyl or hydroxyaminocarbonyl.

In another embodiment of formula 2, n is 6 and R₄Is a hydrogen atom, and R₃Is cyclohexyl. In another embodiment of formula 2, n is 6 and R₄Is a hydrogen atom, and R₃Is methoxy. In another embodiment of formula 2, n is 6, and R is₃And R₄Taken together to form a piperidinyl group. In another embodiment of formula 2, n is 6 and R₄Is a hydrogen atom, and R₃Is benzyloxy. In another embodiment of formula 2, R₄Is a hydrogen atom, and R₃Is gamma-pyridyl. In another embodiment of formula 2, R₄Is a hydrogen atom, and R₃Is beta-pyridyl. In another embodiment of formula 2, R₄Is a hydrogen atom, and R₃Is an alpha-pyridyl group. In another embodiment of formula 2, n is 6, and R is₃And R₄Are both methyl groups. In another embodiment of formula 2, n is 6 and R₄Is methyl, and R₃Is phenyl.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 3, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein n is an integer from 5 to about 8.

In a preferred embodiment of formula 3, n is 6. According to this embodiment, the HDAC inhibitor is SAHA (4) or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or excipient. SAHA can be represented by the following structural formula:

in one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 5, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 6 (pyroxamide), or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 7, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 8, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 9, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 10, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₃Is hydrogen, and R₄Is cycloalkyl, aryl, aryloxy, arylalkoxy or pyridyl, or R₃And R₄Taken together to form a piperidinyl group; r₂Is a hydroxyamino group; and n is an integer from 5 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 11, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₃And R₄Independently substituted or unsubstituted: branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkoxy, aryloxy, arylalkoxy or pyridyl; cycloalkyl, aryl, aryloxy, arylalkoxy or pyridyl, or R₃And R₄Taken together to form a piperidinyl group; r₂Is a hydroxyamino group; and n is an integer from 5 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 12, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently the same or different from each other and are hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino; r is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, an arylalkoxy group, or an aryloxy group; m and n are each independently the same or different from each other and are each an integer of from about 0 to about 8.

In a specific embodiment, the HDAC inhibitor is a compound of formula 12, wherein X, Y and R are each hydroxy, and m and n are both 5.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 13, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently the same or different from each other and are hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino; r₁And R₂Each independently, the same or different from each other, is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, an aryl group, an alkoxy group or an aryloxy group; m, n and o are each independently the same or different from each other and are each an integer of from about 0 to about 8.

In one embodiment of formula 13, X and Y are each hydroxy, and R is₁And R₂Each is methyl. In another embodiment of formula 13, X and Y are each hydroxy, R₁And R₂Each is methyl, n and o are each 6, and m is 2.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 14, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently, the same as or different from each other, hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino; r₁And R₂Each independently, the same or different from each other, is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, an aryl group, an alkoxy group or an aryloxy group; m and n are each independently the same or different from each other and are each an integer of from about 0 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 15, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently, the same as or different from each other, hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino; m and n are each independently the same or different from each other and are each an integer of from about 0 to about 8.

In one embodiment of formula 15, X and Y are each hydroxy, and m and n are each 5.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 16, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently, the same as or different from each other, hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino; r₁And R₂Independently of one another, identical or different and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl group, an arylalkoxy group or an aryloxy group; m and n are each independently the same or different from each other and are each an integer of from about 0 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 17, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently, the same as or different from each other, hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino or aryloxyalkylamino; and n is an integer from about 0 to about 8.

In one embodiment of formula 17, X and Y are each hydroxylamino; r₁Is methyl, R₂Is a hydrogen atom; m and n are each 2. In another specific embodiment of formula 17, X and Y are each hydroxylamino; r₁Is carbonylhydroxyamino, R₂Is a hydrogen atom; m and n are each 5. In another specific embodiment of formula 17, X and Y are each hydroxylamino; r₁And R₂Each is a fluoro group; m and n are each 2.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 18, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein X and Y are each independently, the same as or different from each other, hydroxy, amino or hydroxyamino, substituted or unsubstituted alkoxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino; r₁And R₂Each independently, the same or different from each other, is a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl groupAryl, alkoxy, aryloxy, carbonylhydroxyamino or fluoro; m and n are each independently the same or different from each other and are each an integer of from about 0 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 19, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₁And R₂Independently of one another, identical or different from one another, are hydroxy, alkoxy, amino, hydroxyamino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino. In a specific embodiment, the HDAC inhibitor is a compound of structural formula 19, wherein R₁And R₂Are all hydroxylamino groups.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 20, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₁And R₂Independently of one another, identical or different from one another, are hydroxy, alkoxy, amino, hydroxyamino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino. In a specific embodiment, the HDAC inhibitor is a compound of formula 20, wherein R₁And R₂Are all hydroxylamino groups.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 21, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₁And R₂Independently of one another, identical or different from one another, are hydroxy, alkoxy, amino, hydroxyamino, alkylamino, dialkylamino, arylamino, alkylarylamino, alkoxyamino, aryloxyamino, alkoxyalkylamino or aryloxyalkylamino.

In a specific embodiment, the HDAC inhibitor is a compound of formula 21, wherein R₁And R₂Are all hydroxylamino groups.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 22, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is phenylamino substituted by: cyano, methylcyano, nitro, carboxyl, aminocarbonyl, methylaminocarbonyl, dimethylaminocarbonyl, trifluoromethyl, hydroxyaminocarbonyl, N-hydroxyaminocarbonyl, methoxycarbonyl, chlorine, fluorine, methyl, methoxy, 2, 3-difluoro, 2, 4-difluoro, 2, 5-difluoro, 2, 6-difluoro, 3, 5-difluoro, 2, 3, 6-trifluoro, 2, 4, 6-trifluoro, 1, 2, 3-trifluoro, 3,4, 5-trifluoro, 2, 3,4, 5-tetrafluoro or 2, 3,4, 5, 6-pentafluoro; and n is an integer from 4 to 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor (m-carboxy cinnamic acid dihydroxyamide-CBHA), or a pharmaceutically acceptable salt or hydrate thereof, represented by the structure of formula 23 and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 24, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 25, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is the following substituted or unsubstituted group: phenyl, piperidine, thiazole, 2-pyridine, 3-pyridine or 4-pyridine, and n is an integer from about 4 to about 8.

In one embodiment of formula 25, R is substituted phenyl. In another specific embodiment of formula 25, R is a substituted phenyl group, wherein the substituents are selected from the group consisting of methyl, cyano, nitro, thio (thio), trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2, 3-difluoro, 2, 4-difluoro, 2, 5-difluoro, 3, 4-difluoro, 3, 5-difluoro, 2, 6-difluoro, 1, 2, 3-trifluoro, 2, 3, 6-trifluoro, 2, 4, 6-trifluoro, 3,4, 5-trifluoro, 2, 3,5, 6-tetrafluoro, 2, 3,4, 5, 6-pentafluoro, azido, hexyl, tert-butyl, phenyl, carboxy, hydroxy, methoxy, phenoxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methoxycarbonyl, and phenoxy, Methylaminocarbonyl, dimethylamino, dimethylaminocarbonyl or hydroxyaminocarbonyl.

In another specific embodiment of formula 25, R is a substituted or unsubstituted 2-pyridine, 3-pyridine, or 4-pyridine group, and n is an integer from about 4 to about 8.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 26, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is a substituted or unsubstituted phenyl, pyridine, piperidine, or thiazole group, and n is an integer from about 4 to about 8, or a pharmaceutically acceptable salt thereof.

In one embodiment of formula 26, R is substituted phenyl. In another specific embodiment of formula 26, R is a substituted phenyl group, wherein the substituents are selected from the group consisting of methyl, cyano, nitro, thio, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2, 3-difluoro, 2, 4-difluoro, 2, 5-difluoro, 3, 4-difluoro, 3, 5-difluoro, 2, 6-difluoro, 1, 2, 3-trifluoro, 2, 3, 6-trifluoro, 2, 4, 6-trifluoro, 3,4, 5-trifluoro, 2, 3,5, 6-tetrafluoro, 2, 3,4, 5, 6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxy, hydroxy, methoxy, phenoxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methoxycarbonyl, nitro, thio, trifluoromethyl, amino, aminocarbonyl, methylcyano, 2, 3,4, 5-difluoro, 2, 6-difluoro, 1, 2, 3,4, 6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxy, hydroxy, phenoxy, benzyloxy, Methylaminocarbonyl, dimethylamino, dimethylaminocarbonyl or hydroxyaminocarbonyl.

In another embodiment of formula 26, R is phenyl and n is 5. In another embodiment, n is 5 and R is 3-chlorophenyl.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 27, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₁And R₂Each of which is attached directly or through a linking group and is a substituted or unsubstituted of: aryl (e.g. phenyl), arylalkyl (e.g. benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridylamino, piperidino, 9-purin-6-amino, thiazolylamino, hydroxy, branched or unbranched alkyl, alkenyl, alkoxy, aryloxy, arylalkoxy, pyridyl or quinolinyl or isoquinolinyl; n is an integer of about 3 to about 10, and R₃A group that is a hydroxamic acid, hydroxyamino, hydroxy, amino, alkylamino, or alkoxy group. The linking group may be, for example, an amide moiety, O-, -S-, -NH-, NR₅、-CH₂-、(CH₂)_m-, - (CH ═ CH) -, phenylene, cycloalkylene, or any combination thereof, wherein R is₅Is substituted or unsubstituted C₁-C₅An alkyl group.

In certain embodiments of formula 27, R₁is-NH-R₄Wherein R is₄The following groups substituted or unsubstituted: aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridylamino, piperidino, 9-purin-6-amino, thiazolylamino, hydroxy, branched or unbranched alkyl, alkenyl, alkoxy, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl.

In one embodiment, the methods of the present invention employ an HDAC inhibitor represented by the structure of formula 28, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

Wherein R is₁And R₂Each is a substituted or unsubstituted group of: aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridylamino, piperidino, 9-purin-6-amino, thiazolylamino, hydroxy, branched or unbranched alkyl, alkenyl, alkoxy, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl; r₃A group that is a hydroxamic acid, hydroxyamino, hydroxy, amino, alkylamino, or alkoxy group; r₄Is hydrogen, halogen, phenyl or a cycloalkyl moiety; a, which may be identical or different, represents an amide moiety, O-, -S-, -NH-, NR₅、-CH₂-、-(CH₂)_m-, - (CH ═ CH) -, phenylene, cycloalkylene, or any combination thereof, wherein R is₅Is substituted or unsubstituted C₁-C₅An alkyl group; and n and m are each an integer of 3 to 10.

In yet another embodiment, compounds having a more specific structure within the scope of compounds 27 or 28 are:

in one embodiment, the HDAC inhibitor used in the methods of the present invention is represented by the structure of formula 29:

wherein A is an amide moiety, R₁And R₂Each selected from substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridylamino, 9-purin-6-amino, thiazolylamino, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl; and n is 3-10An integer number.

For example, a compound of formula 29 can have structure 30 or 31:

wherein R is₁、R₂And n has the meaning of formula 29.

In one embodiment, the HDAC inhibitor used in the methods of the present invention is represented by the structure of formula 32:

wherein R is₇Selected from substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridylamino, 9-purin-6-amino, thiazolylamino, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl; n is an integer from 3 to 10, and Y is selected from:

in one embodiment, the HDAC inhibitor used in the methods of the present invention is represented by the structure of formula 33:

wherein n is an integer of 3 to 10 and Y is selected from

And R is₇' is selected from

In one embodiment, the HDAC inhibitor used in the methods of the present invention is represented by the structure of formula 34:

aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridylamino, 9-purin-6-amino, thiazolylamino, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl; n is an integer of 3 to 10, and R₇' is selected from

In one embodiment, the HDAC inhibitor used in the methods of the present invention is represented by the structure of formula 35:

wherein A is an amide moiety, R₁And R₂Each selected from substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridylamino, 9-purin-6-amino, thiazolylamino, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl; r₄Is hydrogen, halogen, phenyl or cycloalkyl moiety, and n is 3-10An integer number.

For example, the compound of formula 35 can have structure 36 or 37:

wherein R is₁、R₂、R₄And n has the meaning of formula 35.

In one embodiment, the HDAC inhibitor used in the methods of the present invention is represented by the structure of formula 38:

wherein L is a linking group selected from: amide moiety, O-, -S-, -NH-, NR₅、-CH₂-、-(CH₂)_m-, - (CH ═ CH) -, phenylene, cycloalkylene, or any combination thereof, wherein R is₅Is substituted or unsubstituted C₁-C₅An alkyl group; and wherein R₇And R₈Each independently substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridylamino, 9-purin-6-amino, thiazolylamino, aryloxy, arylalkoxy, pyridyl, quinolinyl, or isoquinolinyl; n is an integer of 3 to 10, and m is an integer of 0 to 10.

For example, a compound of formula 38 can be represented by the structure of formula (39):

other HDAC inhibitors useful in the methods of the present invention include those shown in the following more specific formulae:

a compound represented by structure (40) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 40, n-5.

A compound represented by structure (41) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 41, n-5.

A compound represented by structure (42) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 42, n-5.

A compound represented by structure (43) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 43, n-5.

A compound represented by structure (44) or an enantiomer thereof:

wherein n is an integer of 3-1, 0. In one specific embodiment of formula 44, n-5.

A compound represented by structure (45) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 45, n-5.

A compound represented by structure (46) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 46, n-5.

A compound represented by structure (47) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 47, n-5.

A compound represented by structure (48) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 48, n-5.

A compound represented by structure (49) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 49, n-5.

A compound represented by structure (50) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 50, n-5.

A compound represented by structure (51) or an enantiomer thereof:

wherein n is an integer of 3 to 10. In one specific embodiment of formula 51, n is 5.

Other examples of such compounds and other HDAC inhibitors may be found in the following documents: U.S. Pat. No. 5,369,108 issued on 11/29/1994, Breslow et al, U.S. Pat. No. 5,700,811 issued on 12/23/1997, U.S. Pat. No. 5,773,474 issued on 30/6/1998, U.S. Pat. No. 5,932,616 issued on 3/8/1999, and U.S. Pat. No. 6,511,990 issued on 28/1/2003, all issued to Breslow et al; U.S. patent No. 5,055,608 issued on day 10/8 of 1991, U.S. patent No. 5,175,191 issued on day 29 of 1992, and U.S. patent No. 5,608,108 issued on day 3/4 of 1997, all issued to Marks et al; and Yoshida, M.et al, Bioassays 17, 423- & 430 (1995); saito, A. et al, PNAS USA 96, 4592-; furamai R. et al, PNAS USA 98(1), 87-92 (2001); komatsu, Y, et al, Cancer Res.61(11), 4459 and 4466 (2001); su, G.H., et al, Cancer Res.60, 3137-; lee, B.I., et al, Cancer Res.61(3), 931-934; suzuki, T, et al, J.Med.chem.42(15), 3001-3003 (1999); PCT application WO 01/18171 to Sloan-Kettering Institute for cancer Research and The Trustees of Columbia University, published 3, 15.2001; 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 5-31-2001), WO 01/70675 (published 9-27-2001) and WO 00/71703 (published 11-30-2000), to methygene, inc; PCT application WO 00/21979 published 10/8 1999 to Fujisawa Pharmaceutical co., ltd; PCT application WO98/40080 published by BeaconLaboratories, l.l.c. on 3/11/1998; and Current patent status of HDAC inhibitors Expert Opin. Ther. patents (2002)12 (9): 1375-.

SAHA or any other HDACs can be synthesized according to the methods described in the experimental section, or according to the methods 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 herein by reference in their entirety, or according to any other method known to those 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 includes any compounds that are structurally similar to the compounds represented below and that are capable of inhibiting histone deacetylases.

Chemical definition

An "aliphatic group" is a non-aromatic group, consisting solely of carbon and hydrogen, and may optionally contain one or more units of unsaturation, such as double and/or triple bonds. Aliphatic groups may be straight chain, branched chain or cyclic. When straight or branched, the aliphatic group typically contains from about 1 to about 12 carbon atoms, more typically from about 1 to about 6 carbon atoms. When cyclic, the aliphatic group typically contains from about 3 to about 10 carbon atoms, more typically from about 3 to about 7 carbon atoms. Preferably the aliphatic radical is C₁-C₁₂Straight or branched alkyl (i.e. fully saturated aliphatic), more preferably C₁-C₆Straight or branched chain alkyl. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl.

As used herein, "aromatic group" (also referred to as "aryl") includes carbocyclic aryl, heterocyclic aryl (also referred to as "heteroaryl") and fused polycyclic aromatic ring systems as defined herein.

A "carbocyclic aryl" is an aromatic ring of 5 to 14 carbon atoms, including carbocyclic aryl fused to a 5 or 6 membered cycloalkyl group such as indane. Examples of carbocyclic aryl groups include, but are not limited to, phenyl, naphthyl such as 1-naphthyl and 2-naphthyl; anthracenyl groups such as 1-anthracenyl group and 2-anthracenyl group; phenanthryl; fluorenone group such as 9-fluorenone group, indanyl group and the like. Optionally, the carbocyclic aryl group is substituted with the indicated number of substituents described below.

A "heterocycloaryl" (or "heteroaryl") group is a monocyclic, bicyclic, or tricyclic aromatic ring of 5 to 14 ring carbon atoms and 1 to 4 heteroatoms selected from O, N or S. Examples of heteroaryl groups include, but are not limited to, pyridyl groups such as 2-pyridyl (also known as α -pyridyl), 3-pyridyl (also known as β -pyridyl), and 4-pyridyl (also known as γ -pyridyl); thienyl such as 2-thienyl and 3-thienyl; furyl groups such as 2-furyl and 3-furyl; pyrimidinyl such as 2-pyrimidinyl and 4-pyrimidinyl; imidazolyl such as 2-imidazolyl; pyranyl groups such as 2-pyranyl and 3-pyranyl; pyrazolyl groups such as 4-pyrazolyl group and 5-pyrazolyl group; thiazolyl such as 2-thiazolyl, 4-thiazolyl and 5-thiazolyl; a thiadiazolyl group; an isothiazolyl group; oxazolyl groups such as 2-oxazolyl, 4-oxazolyl, and 5-oxazolyl; an isoxazolyl group; a pyrrolyl group; a pyridazinyl group; pyrazinyl and the like. The heterocyclic aryl (or heteroaryl) groups defined above may be optionally substituted with a designated number of aryl substituents as described below.

A "fused polycyclic aromatic" ring system is a carbocyclic aryl or heteroaryl group fused to one or more other heteroaryl or non-aromatic heterocyclic rings. Examples include quinolyl and isoquinolyl groups such as 2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl and 8-quinolyl, 1-isoquinolyl, 3-quinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl and 8-isoquinolyl; benzofuranyl groups such as 2-benzofuranyl and 3-benzofuranyl; dibenzofuranyl, for example 2, 3-dihydrobenzofuranyl; a dibenzothienyl group; benzothienyl groups, such as 2-benzothienyl and 3-benzothienyl; indolyl groups, such as 2-indolyl and 3-indolyl; benzothiazolyl groups such as 2-benzothiazolyl group; benzoxazolyl groups such as 2-benzoxazolyl; benzimidazolyl groups such as 2-benzimidazolyl; isoindolyl groups such as 1-isoindolyl and 3-isoindolyl; a benzotriazole group; a purine group; thioindenyl and the like. The polycyclic aromatic ring systems, which may optionally be fused, are substituted with a designated number of substituents described herein.

"arylalkyl" (arylalkyl) is an alkyl group substituted with an aryl group, preferably a phenyl group. A preferred aralkyl group is benzyl. Suitable aryl groups and suitable alkyl groups are described herein. Suitable substituents for aralkyl groups are described herein.

An "aryloxy group" is an aryl group (e.g., phenoxy) attached to a compound through an oxygen.

As used herein, "alkoxy" (alkyloxy) is a straight or branched chain C attached to a compound through an oxygen atom₁-C₁₂Alkyl or cyclic C₃-C₁₂An alkyl group. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, and propoxy.

An "arylalkoxy" (arylalkyloxy) group is an arylalkyl group (e.g., phenylmethoxy) that is attached to the compound through the oxygen on the alkyl portion of the arylalkyl group.

As used herein, "arylamino" is an aryl group attached to a compound through a nitrogen.

As used herein, "arylalkylamino" is an arylalkyl group attached to a compound through a nitrogen on the alkyl portion of the arylalkyl group.

Many of the moieties or groups used herein are referred to as "substituted or unsubstituted". When a moiety is referred to as substituted, it means that any moiety known to those skilled in the art to which the moiety may be substituted. For example, a substitutable group can be a hydrogen atom that is replaced with a group that is not hydrogen (i.e., a substituent). Multiple substituents may be present. When a plurality of substituents are present, the substituents may be the same or different, and substitution may occur at any substitutable position. Such substitution patterns are well known in the art. For illustrative purposes (without intending to limit the scope of the invention), some examples of groups that are substituents are: alkyl (which may also be substituted by one or more substituents, e.g. CF)₃) Alkoxy (which may be substituted, e.g. OCF)₃) Halogen OR halo (F, Cl, Br, I), hydroxy, nitro, oxo, -CN, -COH, -COOH, amino, azido, N-alkylamino OR N, N-dialkylamino wherein the alkyl groups may also be substituted, ester groups (-C (O) -OR wherein R may be groups such as alkyl, aryl, which may be substituted), aryl (most preferably phenyl, which may be substituted), arylalkyl (which may be substituted) and aryloxy.

Stereochemistry

Many organic compounds exist in optically active forms that have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to designate the absolute configuration of the molecule with respect to its chiral center. The symbols for the compound to rotate plane polarized light are denoted by the prefixes d and l or (+) and (-) and the compound is levorotatory by (-) or. Compounds with the prefix (+) or d are dextrorotatory. For a defined chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is commonly referred to as a mixture of enantiomers. A50: 50 mixture of enantiomers is referred to as a racemic mixture. The various compounds described herein may have one or more chiral centers and thus may exist in different enantiomers. If desired, the chiral carbon may be indicated by an asterisk. When in the formula of the present invention the bond to the chiral carbon is drawn as a straight line, it is to be understood that both (R) and (S) configurations of the chiral carbon, and thus the formula includes both enantiomers and mixtures thereof. As used in the art, when it is desired to specify the absolute configuration of a chiral carbon, one of the chiral carbons can be depicted as a wedge (attached to an atom above the plane) and the other as a wedge (attached to an atom below the plane) in dashed or short parallel lines. The (R) or (S) configuration of a chiral carbon can be represented by the Cahn-Inglod-Prelog system.

When the HDAC inhibitors of the present invention contain a chiral center, these compounds exist in two enantiomeric forms, and the present invention includes both enantiomers and mixtures of enantiomers, such as the particular 50: 50 mixture known as a racemic mixture. The compounds can be prepared by methods known to those skilled in the art, for example by forming diastereomeric salts which can be separated; for example by crystallization (see CRC Handbook of Optical resolution via Diastereometric Salt Format by David Kozma (CRC Press, 2001)); formation of diastereomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selectively reacting one enantiomer with an enantiomer-specific reagent, e.g., enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support such as silica gel to which a chiral ligand is bound, or in the presence of a chiral solvent. One skilled in the art will recognize that when the desired enantiomer is converted to another chemical entity by one of the separation methods described above, a further step of liberating the desired enantiomer is required. Alternatively, a particular enantiomer may be synthesized by converting one enantiomer into the other by asymmetric synthesis or by asymmetric transformation using optically active reagents, substrates, catalysts or solvents.

The notation of a particular absolute configuration of a chiral carbon of a compound of the present invention is understood to mean that the designated enantiomeric form of the compound is in enantiomeric excess (ee), or in other words, substantially free of another enantiomer. For example, the "S" form of a compound is substantially free of the "R" form of the compound, and thus is in enantiomeric excess of the "S" form. In contrast, the "R" form of the compound is substantially free of the "S" form of the compound and is therefore an enantiomeric excess of the "R" form. As used herein, enantiomeric excess is the presence of more than 50% of an enantiomer. For example, the enantiomeric excess can be about 60% or more, e.g., about 70% or more, e.g., about 80% or more, e.g., about 90% or more. In particular embodiments, when a particular absolute configuration is indicated, the enantiomeric excess of the compound is at least about 90%. In a more specific embodiment, the compounds have an enantiomeric excess of at least about 95%, such as at least about 97.5%, for example at least 99%.

When the compound of the present invention has two or more chiral carbons, it may have more than two optical isomers, and diastereomers may be present. For example, when there are two chiral carbons, the compound may have up to 4 optical isomers and two pairs of enantiomers ((S, S)/(R, R) and (R, S)/(S, R)). The enantiomeric pairs (e.g., (S, S)/(R, R)) are mirror image stereoisomers of each other. Stereoisomers that are not mirror images (e.g., (S, S) and (R, S)) are diastereomers. Diastereomeric pairs can be separated by methods known to those skilled in the art, such as chromatography or crystallization, and the individual enantiomers in each pair can be separated as described above. The present invention includes each diastereomer of such compounds and mixtures thereof.

As used herein, the expressions and definite articles "the" include both 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 and a combination of two or more different active agents, reference to "a carrier" includes a mixture of two or more carriers and a carrier, and the like.

The invention also includes prodrugs of the HDAC inhibitors disclosed herein. Any prodrug of such compounds may be prepared using well known pharmaceutical techniques.

In addition to the compounds listed above, the present invention also includes the use of homologs and analogs of such compounds. In this context, a homologue is a molecule having a substantially similar structure to the compounds described above, and an analogue is a molecule having substantially biological similarity, whether or not structurally similar.

Alkylating agent

Alkylating agents react with nucleophilic residues such as chemical entities on the nucleotide precursors that generate DNA. They influence the cell division process by alkylating these nucleotides and preventing their assembly into DNA.

Examples of alkylating agents include, but are not limited to, dicloethamine (nitrogen mustards such as chlorambucil, cyclophosphamide, ifosfamide, nitrogen mustards, melphalan, uramustine), aziridines (e.g., thiotepa), alkyl ketosulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, streptozocin), atypical alkylating agents (hexamethylmelamine, dacarbazine, and procarbazine), platinum compounds (carboplatin and cisplatin). These compounds are reacted with phosphate, amino, hydroxyl, mercapto, carboxyl and imidazolyl.

Under physiological conditions, these drugs ionize, producing positively charged ions that attach to nucleic acids and proteins, resulting in cell cycle inhibition and/or cell death. Alkylating agents are cell cycle phase non-specific (phaseonspecific) drugs because they exert their activity independently of the specific phase of the cell cycle. Nitrogen mustards and alkyl ketosulfonates are most effective on cells in either the Gl or M phase. Nitrosoureas, nitrogen mustards, and aziridines affect progression from the Gl and S phases to the M phase. Chabner and Collins, eds (1990) "Cancer Chemotherapy: principles and Practice ", philiadelphia: JB Lippincott.

Alkylating agents are active against a variety of neoplastic diseases and have significant activity in the treatment of leukemias and lymphomas as well as solid tumors. Clinically, such drugs are commonly used to treat acute and chronic leukemia; hodgkin's disease; non-hodgkin lymphoma; multiple myeloma; primary brain tumors; carcinomas of the breast, ovary, testis, lung, bladder, cervix, head and neck, and malignant melanoma.

The main toxicity of common alkylating agents is myelosuppression. In addition, various degrees of gastrointestinal adverse effects generally occur, and various organ toxicities are associated with the specific compounds. Black and Livingston (1990) Drugs 39: 489-; and 39: 652-673.

Antibiotic

Antibiotics (e.g., cytotoxic antibiotics) act by directly inhibiting DNA or RNA synthesis and are therefore effective throughout the cell cycle. Examples of antibiotic drugs include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, and anthracenedione), mitomycin C, bleomycin, actinomycin D, plicatomycin. These antibiotics interfere with cell growth by targeting different cellular components. For example, it is generally believed that anthracyclines interfere with the action of DNA topoisomerase II in regions of transcriptionally active DNA, leading to DNA melting.

Bleomycin is generally thought to chelate iron and form an activated complex, which then binds to the bases of DNA, leading to melting and cell death.

Antibiotic drugs have been used as therapeutic agents for a variety of neoplastic diseases, including carcinomas of the breast, lung, stomach and thyroid, lymphomas, myelogenous leukemias, myelomas and sarcomas. The main toxicity of anthracyclines in this class is myelosuppression, especially granulocytopenia. Mucositis is often accompanied by granulocytopenia, and the severity is related to the degree of myelosuppression. There is also significant cardiotoxicity associated with large dose administration of anthracyclines.

Antimetabolites

Antimetabolites (i.e., antimetabolites) are a class of drugs that interfere with metabolic processes critical to the proliferation and physiology of cancer cells. Actively proliferating cancer cells require the continuous synthesis of large amounts of nucleic acids, proteins, lipids and other important cellular components.

Many antimetabolites inhibit purine or pyrimidine nucleoside synthesis or inhibit DNA replication enzymes. Certain antimetabolites also interfere with ribonucleoside and RNA synthesis and/or amino acid metabolism and protein synthesis. By interfering with the synthesis of important cellular components, antimetabolites can retard or inhibit cancer cell growth. Examples of antimetabolites 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.

Antimetabolites have been widely used to treat several common types of cancer, including cancers of the colon, rectum, breast, liver, stomach, and pancreas, malignant melanoma, acute and chronic leukemia, hairy cell leukemia. Inhibition of cell proliferation in mitotically active tissues such as bone marrow or gastrointestinal mucosa leads to a variety of adverse effects of antimetabolite therapy. Patients treated with these drugs typically experience myelosuppression, stomatitis, diarrhea, and hair loss. Chen and Grem (1992) curr. opin. oncol.4: 1089-1098.

Hormone medicine

Hormonal drugs are a class of drugs that regulate the growth and development of their target organs. Most hormonal drugs are sex steroids and their derivatives and analogues, such as estrogens, progestogens, antiestrogens, androgens, antiandrogens, and progestins. These hormone drugs act as antagonists of sex steroid receptors, down regulating receptor expression and important gene transcription. Examples of such hormonal agents are synthetic estrogens (e.g. diethylstilbestrol), antiestrogens (e.g. tamoxifen, toremifene, fluoxymesterol and raloxifene), antiandrogens (bicalutamide, nilutamide, flutamide), aromatase inhibitors (e.g. aminoglutethimide, anastrozole and tetrazole), Luteinizing Hormone Releasing Hormone (LHRH) analogues, ketoconazole, goserelin acetate, leuprolide, megestrol acetate and mifepristone.

The hormone medicine is used for treating breast cancer, prostatic cancer, melanoma and meningioma. Because the major effects of hormones are mediated through steroid receptors, 60% of receptor-positive breast cancers respond to first-line hormone therapy; and less than 10% of receptor-negative tumors respond. The major side effect associated with hormonal drugs is flushing. The manifestations of the multiple are bone pain, sudden increase in erythema around skin lesions and induction of hypercalcemia.

Progestagens are particularly useful in the treatment of endometrial cancer because these cancers occur in women exposed to high levels of estrogen that the progestagen cannot antagonize.

Antiandrogens are mainly used for the treatment of prostate cancer, and are hormone-dependent. They are used to reduce testosterone levels and thus inhibit tumor growth.

Hormone therapy for breast cancer involves reducing the level of estrogen that is dependent on activating estrogen receptors in breast tumor cells. Antiestrogens act by binding to estrogen receptors and preventing the recruitment of coactivators, thereby inhibiting estrogen signaling.

LHRH analogues are used to treat prostate cancer and reduce testosterone levels and thereby tumor growth.

Aromatase inhibitors act by inhibiting the enzymes required for hormone synthesis. In postmenopausal women, the main source of estrogen is the conversion of androstenedione by aromatase.

Plant-derived medicine

Plant-derived drugs are drugs derived from plants or a class of drugs modified on the basis of the molecular structure of these drugs. They inhibit cell replication by preventing the assembly of cellular components essential for cell division.

Examples of plant-derived drugs include vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), taxanes (e.g., paclitaxel and docetaxel. These plant-derived drugs generally act in the same way as antimitotic drugs, and bind to tubulin to inhibit mitosis. It is believed that podophyllotoxins such as etoposide interfere with DNA synthesis by interacting with topoisomerase II, causing DNA melting.

The plant-derived medicine can be used for treating various cancers. Vincristine is used, for example, in the treatment of leukemia, hodgkin and non-hodgkin lymphomas and childhood tumors neuroblastoma, rhabdomyosarcoma and wilms' tumor. Vinblastine is used for treating lymphoma, testis cancer, renal cell carcinoma, mycosis fungoides and Kaposi sarcoma. Docetaxel has been shown to be expected to have activity against advanced breast cancer, non-small cell lung cancer (NSCLC) and ovarian cancer.

Etoposide has activity against a variety of tumors, of which small cell lung cancer, testicular cancer, and NSCLC are the most sensitive.

Plant-derived drugs cause significant side effects in the treated patients. Vinca alkaloids exhibit different clinical toxicities. Side effects of vinca alkaloids include neurotoxicity, altered platelet function, bone marrow suppression, and leukopenia. Paclitaxel causes dose-limiting neutropenia and a relative deficiency in other hematopoietic cell lines. The major toxicity of podophyllotoxin is hematologic toxicity (neutropenia and thrombocytopenia).

Other side effects include transient liver enzyme abnormalities, hair loss, allergic reactions and peripheral neuropathy.

Biological medicine

Biopharmaceuticals are a class of biomolecules that cause cancer/tumor regression when used alone or in combination with chemotherapy and/or radiotherapy. Examples of biological agents include immunomodulatory proteins such as cytokines, monoclonal antibodies against tumor antigens, tumor suppressor genes, and cancer vaccines.

Cytokines have extremely potent immunomodulatory activity. Certain cytokines such as interleukin-2 (IL-2, aldesleukin) and alpha-interferon (IFN- α) demonstrate anti-tumor activity and are approved for the treatment of patients with metastatic renal cell carcinoma and metastatic malignant melanoma. IL-2 is a T cell growth factor important for T cell-mediated immune responses. It is believed that the selective anti-tumor effects of IL-2 in certain patients are the result of a cell-mediated, discriminatory immune response between self and non-self.

Interferon-alpha includes more than 23 related subtypes with additive activity. IFN- α demonstrates activity against a variety of solid and hematologic malignancies, the latter of which appears to be particularly sensitive.

Examples of interferons include alpha-interferon, beta-interferon (fibroblast interferon), and gamma-interferon (fibroblast interferon). Examples of other cytokines include erythropoietin (α -erythropoietin), granulocyte-CSF (filgrastim), and granulocyte, macrophage-CSF (sargrastim). Other non-cytokine immunomodulators include bacillus Calmette-Guerin, levamisole and octreotide, long-acting octapeptides that act like the naturally occurring hormone somatostatin.

In addition, anti-cancer treatment may include treatment by immunotherapy using antibodies and agents used in tumor vaccination methods. The main drugs in such therapies are antibodies alone or carrying drugs such as toxins or chemotherapeutic drugs/cytotoxic drugs to cancer cells. Monoclonal antibodies against tumor antigens are antibodies which elicit an antigen expressed against a tumor, preferablyA tumor specific antigen. For example, monoclonal antibodies have been proposed(trastuzumab) against human epidermal growth factor receptor 2(HER2) which is overexpressed in certain breast tumors, including metastatic breast cancer. Overexpression of the HER2 protein is associated with a more aggressive disease and a poorer clinical prognosis.Alone for the treatment of metastatic breast cancer patients with tumors overexpressing HER2 protein.

Another example of a monoclonal antibody against a tumor antigen is(rituximab) which was proposed to combat CD20 on lymphoma cells and to selectively deplete normal and malignant CD20+ pre-B cells and mature B cells.

RITUXAN alone is used to treat patients with relapsed or refractory low grade or follicular, CD20+ B cell non-hodgkin lymphoma.(Gimumab ozomicin) and(alemtuzumab) is yet another example of a monoclonal antibody against a tumor antigen that can be used.

Tumor suppressor genes are genes that act to inhibit the cell growth and division cycle, thus preventing tumor development. Tumor suppressor gene mutations cause cells to ignore one or more inhibitory signal network components, overcome cell cycle checkpoints, and result in the control of higher rates of cell growth-cancer. Examples of tumor suppressor genes include Duc-4, NF-1, NF-2, RB, p53, WT1, BRCA1, and BRCA 2.

DPC4 is involved in pancreatic cancer and is involved in the cytosolic pathway that inhibits cell division. NF-1 encodes a protein that inhibits Ras, which is a cytoplasmic inhibitory protein. NF-1 is involved in neurofibromas and pheochromocytomas of the nervous system and myeloid leukemia. NF-2 encodes nuclear proteins involved in meningiomas, schwannomas and ependymomas of the nervous system. RB encodes a pRB protein, which is the major inhibitor nucleoprotein of the cell cycle. RB is involved in retinoblastoma and bone, bladder, small cell lung and breast cancer. P53 encodes a P53 protein that regulates cell division and induces apoptosis. P53 was found to be mutated and/or inactivated in various cancers. WTI is involved in renal wilms' tumor. BRCA1 is implicated in breast and ovarian cancers, and BRCA2 is implicated in breast cancer. Tumor suppressor genes can be transferred into tumor cells in which they can exert their tumor suppressing function.

Cancer vaccines are a class of drugs that induce the body to produce a specific immune response to tumors. Most cancer vaccines in research, development and clinical trials are Tumor Associated Antigens (TAAs). TAAs are structures (i.e., proteins, enzymes, or carbohydrates) that are present on tumor cells and are relatively absent or reduced on normal cells. Since they are quite unique to tumor cells, TAAs provide targets for the immune system to recognize and cause their destruction. Examples of TAAs include gangliosides (GM2), Prostate Specific Antigen (PSA), Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA) (produced by colon and other adenocarcinomas such as breast, lung, stomach and pancreatic cancers), melanoma-associated antigens (MART-1, gap100, MAGE 1, 3 tyrosinase), papillomavirus E6 and E7 fragments, whole cells or parts/lysates of autologous and heterologous tumor cells.

Other therapies

In addition to the treatment of cancer with traditional cytotoxic and hormonal therapies, other therapies that have been newly developed to treat cancer have also been introduced.

For example, various forms of gene therapy are in preclinical or clinical trials.

In addition, methods based on inhibition of tumor angiogenesis (vasculogenesis) are currently being developed. The purpose of this concept is to cut off the tumor nutrient and oxygen supply provided by the newly formed tumor vasculature.

In addition, cancer therapy by inducing terminal differentiation of tumor cells has also been attempted. Suitable differentiating agents include compounds disclosed in any one or more of the following references, the contents of which are incorporated herein by reference.

b) derivatives of vitamin D and tretinoin (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 while 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-;

c) steroid hormones (Lotem, j. and Sachs, L. (1975) int.j. cancer 15: 731-740);

g) inhibitors of DNA or RNA synthesis (Schwartz, e.l., and Sartorelli, a.c. (1982) Cancer res.42: 2651-: 2795-; 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 sartorlli, a.c. (1983) Cancer res.43: 2725-; sugano, h., Furusawa, m., Kawaguchi, t., and Ikawa, Y. (1973) bibl.hematol.39: 943-954; ebert, p.s., Wars, i., and Buell, D.N, (1976) cancer res.36: 1809-1813; hayashi, m., Okabe, j., and Hozumi, m. (1979) Gann 70: 235-238),

the use of all these methods in combination with HDAC inhibitors such as SAHA is within the scope of the present invention.

Modes of administration and dosages

The methods of the invention comprise administering to a subject in need thereof a first amount of an HDAC inhibitor, e.g., SAHA, in a first treatment regimen, and a second amount of an anti-cancer agent in a second treatment regimen. The first and second treatments together comprise a therapeutically effective amount.

The term "patient" as used herein refers to a recipient of treatment. Including mammalian and non-mammalian patients. In particular embodiments, the patient is a mammal, such as a human, canine, murine, feline, bovine, ovine, porcine, or caprine. In a specific embodiment, the patient is a human.

HDAC inhibitor administration

Route of administration

The HDAC inhibitor (e.g., SAHA) can be administered by any method of administration known to those skilled in the art. Examples of routes of administration include, but are not limited to, oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, liposomal; by inhalation, vaginal, intraocular; local release through a catheter or impression, subcutaneous, intralipid, intraarticular, intrathecal, or sustained release dosage forms.

For example, the HDAC inhibitor of the present 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 inhibitor may be administered intravenously (single bolus or infusion), intraperitoneally, subcutaneously, or intramuscularly, all using forms well known to those of ordinary skill in the pharmaceutical arts. The currently preferred form of administration of the HDAC inhibitor is oral.

The HDAC inhibitor may also be administered in a long acting injectable formulation or an implantable formulation, which may be formulated in a manner that allows for the slow release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders for subcutaneous or intramuscular implantation as a long acting injection or implant. The implant may be of an inert material such as a biodegradable polymer or synthetic silicone, for example silicone rubber, silicone rubber or other polymers produced by the Dow-Corning Corporation.

HDAC inhibitors may also be administered in liposome delivery systems such as small unilamellar liposomes (small unilamellar liposomes), large unilamellar liposomes (large unilamellar liposomes) and multilamellar liposomes (multilamellar vesicles). Liposomes can be formed from various phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

The HDAC inhibitor may also be released using a monoclonal antibody as a separate carrier that binds to the compound molecule.

HDAC inhibitors can also be prepared using soluble polymers as targeting drug carriers. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethyl-asparagine-phenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. In addition, HDAC inhibitors can be prepared with biodegradable polymers for achieving controlled release of drugs, such as polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphiphilic block copolymers of hydrogels.

In a currently preferred embodiment, the HDAC inhibitor, e.g., SAHA, is administered orally in a gelatin capsule, which may contain excipients such as microcrystalline cellulose, croscarmellose sodium, and magnesium stearate. A further preferred embodiment is a gelatin capsule containing 200mg of solid SAHA and 89.5mg of microcrystalline cellulose, 9mg of croscarmellose sodium and 1.5mg of magnesium stearate.

Dosage and dosage regimen

The dosage regimen for using the HDAC inhibitor can be selected based on a variety of factors, including the species, age, weight, sex, and type of cancer being treated; the severity (i.e., stage) of the cancer being treated; the route of administration; renal and hepatic function of the patient; and the specific compound or salt thereof used. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, e.g., prevent, inhibit (completely or partially), or arrest the progress of the disease.

For example, SAHA or any of the HDAC inhibitors may be administered in a total daily dose of up to 800 mg. The HDAC inhibitor can be administered once a day (QD), or divided into multiple daily doses, e.g., twice a day (BID) and three times a day (TID). The HDAC inhibitor may be administered in a total daily dose of up to 800mg, for example 200mg, 300mg, 400mg, 600mg or 800mg, which may be administered once a day or may be divided into multiple daily doses as described above. Oral administration is preferred.

In addition, administration may be continuous, i.e., daily, or intermittent. The term "intermittent" as used herein means stopping and starting at fixed or unfixed intervals. For example, the HDAC inhibitor may be administered intermittently between 1-6 days per week, or it may mean periodic administration (e.g., daily for 2-8 consecutive weeks followed by a rest period of up to 1 week) or it may mean alternate-day administration.

Can be added according to the proportion of 25-4000mg/m²The patient is administered a total daily dose of SAHA or any HDAC inhibitor. Presently preferred treatment regimens include administration of a total daily dose of about 200mg to about 600mg once, twice or three times daily in a row.

Another presently preferred treatment regimen comprises intermittent administration of a total daily dose of about 200mg to about 600mg once, twice or three times daily for 3-5 days per week.

In a specific embodiment, the HDAC inhibitor is administered once daily at a dose of 400mg, or twice daily at a dose of 200 mg.

In another specific embodiment, the HDAC inhibitor is administered intermittently at a dose of 400mg, once daily, or twice daily at a dose of 200mg for three days per week.

In another specific embodiment, the HDAC inhibitor is administered intermittently at a dose of 400mg, once daily, or twice daily at a dose of 200mg, four days per week.

In another specific embodiment, the HDAC inhibitor is administered intermittently at a dose of 400mg, once daily, or twice daily at a dose of 200mg, five days per week.

In a specific embodiment, the HDAC inhibitor is administered once daily at a dose of 600mg, twice daily at a dose of 300mg, or three times daily at a dose of 200 mg.

In another specific embodiment, the HDAC inhibitor is administered intermittently at a dose of 600mg once daily, at a dose of 300mg twice daily, or at a dose of 200mg three times daily, three days per week.

In another specific embodiment, the HDAC inhibitor is administered once daily at a dose of 600mg, twice daily at a dose of 300mg, or three times daily at a dose of 200mg, intermittently over four days per week.

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

In addition, the HDAC inhibitor may be administered for several weeks following any of the regimens described above, followed by a drug withdrawal period. For example, the HDAC inhibitor may be administered for 2-8 weeks followed by a one-week rest period, or as a 300mg dose twice daily for 3-5 days according to any of the above regimens. In another specific embodiment, the HDAC inhibitor is administered three times daily for two consecutive weeks, followed by one week off.

The patient is receiving intravenously or subcutaneously a sufficient amount of the drug to deliver about 3-1500mg/m²Per day, e.g., about 3, 30, 60, 90, 180, 300, 600, 900, 1200 or 1500mg/m²(ii) a daily amount of an HDAC inhibitor. Such amounts may be administered in a variety of suitable ways, for example, by administering large volumes of low concentrations of the HDAC inhibitor over an extended period of time or once or several times a day. These amounts may be administered weekly (7 days) for one or more consecutive days, on an intermittent day, or a combination thereof. Alternatively, a small volume of a high concentration of an HDAC inhibitor is administered once a day for a short period of time, e.g., one or more days continuously, intermittently, or a combination thereof every week (7 days). For example, each treatment may be carried out at 300mg/m for 5 consecutive days²Daily dose administration of 1500mg/m². In another dosing regimen, the number of consecutive days may also be 5 days, the treatment lasts for 2 or 3 consecutive weeks, and 3000mg/m of total treatment is given²And 4500mg/m²。

Generally, intravenous formulations containing HDAC inhibitors at concentrations of about 1.0mg/mL to about 10mg/mL, such as 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL, 7.0mg/mL, 8.0mg/mL, 9.0mg/mL, and 10mg/mL, and administered in amounts to achieve the above dosages, can be prepared. In one example, a patient may be administered a day a sufficient volume of intravenous formulation such that the total daily dose is from about 300 to about 1500mg/m²。

Preferably, the subcutaneous formulations are prepared at a pH of about 5 to about 12 according to methods well known in the art and also include suitable buffering agents and isotonic agents as described below. They may be formulated to release a daily dose of the HDAC inhibitor by subcutaneous administration once or more times daily, e.g., once, twice or three times daily.

The HDAC inhibitor may also be administered by topical application of a suitable intranasal vehicle or by transdermal route using transdermal patches well known to those of ordinary skill in the art. For administration in the form of a transdermal delivery system, the dosage administered is naturally continuous rather than intermittent throughout the dosage regimen.

It will be apparent to those skilled in the art that the various modes of administration, dosages and regimens described herein are illustrative of specific embodiments only and are not intended to limit the broad scope of the invention. Any permutation, variation and combination of dosages and dosing regimens is included within the scope of the invention.

Administration of anticancer drugs

Any one or more specific doses and dosage regimens of the HDAC inhibitor are also applicable to any one or more anti-cancer agents used in combination therapy.

In addition, the specific dosage and dosage regimen of the anti-cancer agent may also vary, and the optimal dosage, dosage regimen, and route of administration will depend on the particular anti-cancer agent being used.

Naturally, the route of administration of SAHA or any other HDAC inhibitor is independent of the route of administration of the anti-cancer agent. The presently preferred route of administration of SAHA is oral. Thus, according to this embodiment, SAHA is administered orally, and the second drug (anti-cancer drug) can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, buccally, intranasally, as liposomes; by inhalation, vaginal, intraocular; local delivery through a catheter or impression, subcutaneous, intralipid, intraarticular, intrathecal, or in a sustained release dosage form.

Furthermore, the HDAC inhibitor and the anti-cancer agent can be administered by the same mode of administration, i.e., for example, orally, IV administration of both agents. However, it is also within the scope of the invention to administer the HDAC inhibitor via one mode of administration, e.g., orally, and the anti-cancer agent via another mode of administration, e.g., IV or any one or more of the other modes of administration described above.

Commonly administered anticancer agents and daily doses include, but are not limited to:

an antimetabolite: 1. methotrexate 20-40mg/m² i.v.

4-6mg/m² p.o.

12000mg/m²High dose therapy

2.6-mercaptopurine 100mg/m²

3.6-thioguanine 1-2X 80mg/m² p.o.

4. Pentostatin 4mg/m² i.v.

5. Fludarabine phosphate 25mg/m² i.v.

6. Cladribine 0.14mg/kg body weight i.v.

7.5-Fluorouracil 500-2600mg/m² i.v.

8. Capecitabine 1250mg/m² p.o.

9. Cytarabine 200mg/m² i.v.

3000mg/m²i.v. high dose therapy

10. Gemcitabine 800-1250mg/m² i.v.

11. Hydroxyurea 800-4000mg/m² p.o.

Antibiotics: 12. actinomycin D0.6 mg/m² i.v.

13. Daunorubicin 45-6.0mg/m² i.v.

14. Doxorubicin 45-60mg/m² i.v.

15. Epirubicin 60-80mg/m² i.v.

16. Idarubicin 10-12mg/m² i.v.

35-50mg/m² p.o.

17. Mitoxantrone 10-12mg/m² i.v.

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

19. Mitomycin C10-20 mg-² i.v.

20. Irinotecan 350mg/m² i.v.

(CPT-11)

21. Topotecan 1.5mg/m² i.v.

An alkylating agent: 22. 6mg/m nitrogen mustard² i.v.

23. Estramustine phosphate 150-200mg/m² i.v.

480-550mg/m² p.o.

24. Melphalan 8-10mg/m2 i.v.

15mg/m² i.v.

25. Chlorambucil 3-6mg/m² i.v.

26. 40-100mg/m of prednimustine² p.o.

27. Cyclophosphamide 750 ion concentration 1200mg/m² i.v.

50-100mg/m² p.o.

28. Ifosfamide 1500-2000mg/m² i.v.

29. 25-200mg/m of trofosfamide² p.o.

30. Busulfan 2-6mg/m² p.o.

31. Trioshusufan 5000-grade acetone-sodium succinate 8000mg/m² i.v.

750-1500mg/m² p.o.

32. Thiotepa 12-16mg/m² i.v.

33. Carmustine (BCNU) 100mg/m² i.v.

34. Lomustine (CCNU) 100-130mg/m² p.o.

35. Nimustine (ACNU) 90-100mg/m² i.v.

36. Dacarbazine (OTIC) 100-375mg/m² i.v.

37. Procarbazine 100mg/m² p.o.

38. Cisplatin 20-120mg/m² i.v.

39. 300mg/m of carboplatin² i.v.

Antimitotic agents: 40. vincristine 1.5-2mg/m² i.v.

41. Vinblastine 4-8mg/m² i.v.

42. Vinca amide 2-3mg/m² i.v.

43. Etoposide (VP16) 100-200mg/m² i.v.

100mg p.o

44. Teniposide (VM26) 20-30mg/m² i.v.

45. Paclitaxel (taxol) 175-250mg/m² i.v.

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

Hormones, cytokines and vitamins

Element: 47. alpha-interferon 2-10×10⁶IU/m²

48. Prednisone 40-100mg/m² p.o.

49. Dexamethasone 8-24mg p.o.

S.c G-CSF 5-20 μ G/kg body weight.

aI/-trans retinoic acid 45mg/m²

52. Interleukin-218X 10⁶IU/m²

53.GM-CSF 250mg/m²

54. Erythropoietin 150IU/kg tiw

Combined administration

The first treatment regimen of administering the HDAC inhibitor can be performed prior to the second treatment regimen, i.e., the anti-cancer agent, after treatment with the anti-cancer agent, concurrently with the anti-cancer agent, or a combination thereof. For example, the total course of therapy for the HDAC inhibitor can be determined. The anti-cancer agent may be administered before the start of treatment with the inhibitor or after treatment with the inhibitor. Furthermore, the anti-cancer drug treatment may be administered during the administration of the inhibitor, but need not occur during the entire inhibitor treatment.

SAHA or any of the HDAC inhibitors can be administered at any dose and dosing regimen in combination with the anti-cancer drug effect to achieve a dose effective to treat cancer.

Pharmaceutical composition

As described above, the HDAC inhibitor and/or anticancer agent-containing composition may be formulated into any dosage form suitable for administration by the following routes: oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, liposomal; by inhalation, vaginal or intraocular administration; local release administration through a catheter or an impression, or subcutaneous, intralipid, intra-articular cavity, intrathecal administration, or administration in a sustained release formulation.

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

The invention also includes pharmaceutical compositions comprising pharmaceutically acceptable salts of HDAC inhibitors and/or anti-cancer agents. Suitable and pharmaceutically acceptable salts of the compounds described herein that are suitable for use in the methods of the present invention can include base or acid addition salts such as salts of inorganic base salts, e.g., alkali metal salts (e.g., lithium, sodium, potassium, etc.), alkaline earth metal salts (e.g., calcium, magnesium, etc.), ammonium salts; organic base salts such as organic amine salts (e.g., triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, ethylenediamine N, N' -dibenzyl salt, etc.), and the like; inorganic acid addition salts (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.); organic carboxylic or sulfonic acid addition salts (e.g., formates, acetates, trifluoroacetates, maleates, tartrates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, and the like); salts of basic or acidic amino acids (e.g., arginine, aspartic acid, glutamic acid, etc.), and the like.

The invention also includes pharmaceutical compositions comprising hydrates of HDAC inhibitors and/or anti-cancer agents. The term "hydrate" includes, but is not limited to, hemihydrate, monohydrate, dihydrate, trihydrate and the like.

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

For oral administration, the pharmaceutical composition may 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 commonly used as a carrier or diluent, such as gums, starches, sugars, cellulosic materials, acrylates, or mixtures thereof, may be used in the formulations of the present invention. The composition may further comprise a disintegrant and a lubricant, and in addition, may comprise one or more additives selected from the group consisting of: a binder, a buffer, a protease inhibitor, a surfactant, a solubilizer, a plasticizer, an emulsifier, a stabilizer, a viscosity enhancer, a sweetener, a film former, or any combination thereof. In addition, the compositions of the present invention may be in a controlled release or immediate release formulation.

The HDAC inhibitor as an active ingredient may be administered in admixture with a suitable pharmaceutical diluent, excipient or carrier (collectively referred to herein as "carrier" material or "pharmaceutically acceptable carrier") suitably selected in accordance with the intended form of administration. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Suitable carriers are described in standard reference texts in the art, the latest edition Remington's pharmaceutical Sciences, which is incorporated herein by reference.

For liquid formulations, the pharmaceutically acceptable carrier may be an aqueous or non-aqueous solution, suspension, emulsion or oil. 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, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil and cod liver oil. The solution or suspension may also include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with an acid or base 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, its use in the compositions is acceptable. Auxiliary active compounds may also be added to the composition.

Solid carriers/diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, glucose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethacrylates), carbon carbonate, magnesium oxide, talc, or mixtures thereof.

In addition, the compositions may contain binders (e.g., acacia, corn starch, gelatin, carbomer, ethylcellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silica, croscarmellose sodium, cross-linked polyvinylpyrrolidone, guar gum, sodium starch glycolate, sodium carboxymethyl starch (Primogel)), buffers of various pH and ionic strength (e.g., tris-HCl, acetate, phosphate), additives to retard surface adsorption such as albumin or gelatin, detergents (e.g., Tween 20, Tween 80, poloxamer F68, cholate), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), penetration enhancers, solubilizers (e.g., glycerol, polyethylene glycol), glidants (e.g., colloidal silica), Antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropylcellulose, hydroxypropylmethylcellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavor), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), glidants (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymeric coating agents (e.g., poloxamers or ethylenediamine derivatives thereof (poloxamines)), coating agents and film forming agents (e.g., ethylcellulose, hydroxypropylcellulose, sodium lauryl sulfate), film forming agents (e.g., hydroxypropylcellulose, sodium laurylami, Acrylates, polymethacrylates) and/or auxiliaries.

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

Oral compositions in dosage unit form are particularly preferred for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units of unit dosage suitable for use in the treatment of a patient; each unit containing a predetermined quantity of active compound expected 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 compounds, the particular therapeutic effect to be achieved, and the inherent limitations of the art of combining such active compounds for the treatment of individuals.

The pharmaceutical composition may be included in a container, package or dispenser together with instructions for administration.

The preparation of pharmaceutical compositions containing the active ingredients is well understood in the art, for example, by mixing, granulating or tablet-forming methods. The active therapeutic ingredient is typically mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. The active drugs for oral administration are mixed with additives usually employed for this purpose, such as vehicles, stabilizers or inert diluents, and converted by customary methods into suitable administration forms, such as tablets, coated tablets, hard or soft gelatin capsules as described in detail above; water, alcohol or oil solutions, and the like.

The amount of compound administered to the patient is less than the toxic amount of the patient. In certain embodiments, the amount of the compound administered to the patient is less than an amount that results in a concentration of the compound in the patient's plasma that equals or exceeds the toxic level of the compound. Preferably, 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 plasma of the patient is maintained at about 25 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 50 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 100 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 500 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 1000 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 2500 nM. In another embodiment, the concentration of the compound in the plasma of the patient is maintained at about 5000 nM. Using HMBA, it was found that the amount of compound administered was about 5g/m²Daily to about 30g/m²A day, in particular about 20g/m²Effective daily without toxicity in the patient. In the practice of the present invention, the optimal amount of compound to be administered to a patient will depend on the particular compound used and the type of cancer being treated.

The percentages of active ingredient and various excipients in the formulation may vary. For example, the composition may contain 20-90%, preferably 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 moderate buffering capacity within a pH range acceptable for intravenous administration can be used as a buffering agent. It is also possible to use sodium chloride solutions in which the pH has been adjusted to the desired range with acids or bases, such as hydrochloric acid or sodium hydroxide. Generally, the pH of an intravenously administered formulation may range from about 5 to about 12. A preferred pH range for an intravenously administered formulation containing an HDAC inhibitor, wherein the HDAC inhibitor has a hydroxamic acid moiety, can be from about 9 to about 12.

Subcutaneous formulations are preferably prepared at a pH in the range of about 5 to about 12, which also contain suitable buffering agents and isotonicity agents, according to methods well known in the art. They may be formulated to deliver a daily dose of the active agent by subcutaneous administration one or more times daily. The selection of the appropriate buffer and pH of the formulation depends on the solubility of the HDAC inhibitor to be administered, and can be readily made by one of ordinary skill in the art. Sodium chloride solutions in which the pH has been adjusted to the desired range with acids or bases such as hydrochloric acid or sodium hydroxide may also be used in subcutaneous formulations. Typically, the pH of the subcutaneous formulation may range from about 5 to about 12. The preferred pH range for subcutaneous formulations of HDAC inhibitors with hydroxamic acid moieties can be from about 9 to about 12.

The compositions of the present invention may also be administered in intranasal form by topical use of suitable intranasal vehicles or by transdermal routes, using those forms of transdermal patches well known to those of ordinary skill in the art. For administration in the form of a transdermal delivery system, the dosage administered is naturally continuous rather than intermittent throughout the dosage regimen.

The present invention also provides a method for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in vitro by contacting such cells with a first amount of suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof and a second amount of an anti-cancer agent, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest, apoptosis, and thereby inhibit proliferation of such cells.

Although the methods of the invention may be practiced in vitro, it is contemplated that preferred embodiments of the methods of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells will involve contacting the cells in vivo, i.e., by administering these compounds to a patient in need of treatment having latent neoplastic or tumor cells.

Accordingly, the present invention also provides a method of 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 regimen, and a second amount of an anti-cancer agent in a second treatment regimen, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest, apoptosis of the cells.

In the experimental details which follow, the invention is illustrated by way of example. This section is described to aid in understanding the invention, but is not intended to, nor in any way, limit the invention described in the claims.

Detailed description of the experiments section

Example 1:

synthesis of SAHA

SAHA can be synthesized as described below, or as described in U.S. patent No. 5,369,108, which is incorporated herein by reference in its entirety, or by any other method.

Synthesis of SAHA

Step 1-Synthesis of suberic acid monoacylanilide (Suberanic acid)

In a 22L flask, 3,500g (20.09mol) of suberic acid was added, and the acid was melted by heating. The temperature was raised to 175 ℃ and then 2,040g (21.92mol) of aniline were added. The temperature was raised to 190 ℃ and maintained at this temperature for 20 minutes. The melt was poured into a Nalgene tank containing 4,017g of a 50L aqueous solution of potassium hydroxide. The mixture was stirred for 20 minutes, followed by addition of the melt. The reaction was repeated on the same scale and a second batch of melt was poured into the same potassium hydroxide solution. After the mixture was sufficiently stirred, the stirrer was turned off, and the mixture was allowed to settle. The mixture was then filtered through a pad of celite (4,200g) (the product was filtered to remove neutral by-products (produced by the chemical reaction of aniline at both ends of suberic acid.) the filtrate contained the salt of the product and the salt of unreacted suberic acid. Acidifying the filtrate with 5L concentrated hydrochloric acid; the mixture was stirred for 1 hour and then allowed to settle overnight. The product was filtered, collected and washed on the funnel with deionized water (4X 5L). The wet cake was placed in a 72L flask containing 44L deionized water, the mixture was heated to 50 deg.C, the solids were isolated by hot filtration (the desired product was contaminated with much more soluble suberic acid in hot water. several hot triturations removed suberic acid. NMR [ D₆DMSO]The product was checked to monitor the removal of suberic acid). The hot trituration was repeated with 44L of water at 50 ℃. The product was again isolated by filtration and rinsed with 4L of hot water. Drying at 65 deg.C over weekend in a vacuum drying oven using a Nash pump (Nash pump is a liquid (water) ring pump that pumps up to about 29 inches of mercury, intermittently filled with argon to aid in the delivery of water); 4,182.8g of suberic monoacylanilide are obtained.

The product still contained a small amount of suberic acid; thus, hot milling was carried out at 65 ℃ in portions with about 300g of product at a time. Each portion was filtered and rinsed thoroughly with hot water (about 6L total). The process was repeated to purify the entire batch of product. This completely removed the suberic acid from the product. The solid products were combined in a flask, stirred with 6L methanol/water (1: 2), then isolated by filtration, and air dried on the filter over the weekend. They were placed in trays and dried at 65 ℃ for 45 hours in a vacuum oven using a Nash pump and a stream of argon. The weight of the final product was 3,278.4g (32.7% yield).

Step 2-Synthesis of suberic acid-Acylanilide Methyl ester (Methyl Suberanilate)

A50L flask equipped with a mechanical stirrer and condenser was charged with 3,229g of suberic monoacrylanilide from the previous step, 20L of methanol and 398.7g of Dowex 50WX2-400 resin. The mixture was heated to reflux and maintained at reflux for 18 hours. The mixture was filtered to remove the resin beads and the filtrate was evaporated to residue on a rotary evaporator.

The residue from the rotary evaporator was transferred to a 50L flask equipped with a mechanical stirrer and a condenser. To the flask was added 6L of methanol, and the mixture was heated to give a solution. Then 2L of deionized water was added and the heating was stopped. The mixture was cooled with stirring, and then the flask was placed in an ice bath to cool the mixture. The solid product was isolated by filtration and the filter cake was rinsed with 4L of cold methanol/water (1: 1). The product was dried at 45 ℃ for a total of 64 hours in a vacuum oven using a Nash pump to give 2,850.2g (84% yield) suberic acid-anilide methyl ester, CSL run No. 98-794-92-31.

Step 3-Synthesis of crude SAHA

A50L flask equipped with a mechanical stirrer, thermocouple, and inert gas cannula was charged with 1,451.9g of hydroxylamine hydrochloride, 19L of anhydrous methanol, and 3.93L of 30% sodium methoxide in methanol. 2,748.0g of suberic acid-anilide methyl ester were then added to the flask, followed by 1.9L of 30% sodium methoxide in methanol. The mixture was stirred for 16 hours and 10 minutes. About half of the reaction mixture in the reaction flask (flask 1) was transferred to a 50L flask (flask 2) equipped with a mechanical stirrer. Then 27L of deionized water was added to flask 1 and the mixture was stirred for 10 minutes. Measuring the pH with a pH meter; the pH was 11.56. The pH of the mixture was adjusted to 12.02 by adding 100ml of 30% sodium methoxide in methanol; a clear solution was obtained (at which point the reaction mixture contained a small amount of solid. pH was adjusted to give a clear solution from which the product precipitated). The reaction mixture in flask 2 was diluted in the same way; 27L of deionized water was added, and 100ml of 30% sodium methoxide solution was added to the mixture to adjust the pH, resulting in a pH of 12.01 (clear solution).

The reaction mixture in each flask was acidified by the addition of glacial acetic acid and the product precipitated. The final pH of flask 1 was 8.98 and the final pH of flask 2 was 8.70. The product in both flasks was isolated by filtration using a buchner funnel and filter cloth. The filter cake was washed with 15L of deionized water, the funnel was covered and the product on the funnel was partially dried under vacuum for 15.5 h. The product was removed and placed in 5 glass trays. The trays were placed in a vacuum oven and the product was dried to constant weight. The first drying period was 22 hours at 60 ℃ with a Nash pump as a vacuum source and a stream of argon. The pan was removed from the vacuum oven and weighed. The tray was returned to the drying cabinet and the product was dried for 4h and 10 min using an oil pump as the vacuum source without argon flow. The material was packaged in a double layer 4-mil polyethylene bag and placed in a plastic-shelled container. After sampling, the final weight was 2633.4g (95.6%).

Step 4-recrystallization of crude SAHA

The crude SAHA was recrystallized in methanol/water. A50L flask equipped with a mechanical stirrer, thermocouple, condenser and inert gas cannula was charged with the crude SAHA to be crystallized (2,525.7g), followed by 2,625ml deionized water and 15,755ml methanol. The material was heated to reflux to give a solution. 5,250ml of deionized water was then added to the reaction mixture. The heating was stopped and the mixture was allowed to cool. When the mixture was cold enough (28 ℃) to safely dispose of the flask, the flask was removed from the heating mantle and placed in a bathtub used as a cold bath. Ice/water was added to the tub and the mixture was cooled to-5 ℃. The mixture was kept below this temperature for 2 hours. The product was isolated by filtration and the filter cake was washed with 1.5L of cold methanol/water (2: 1). The funnel was masked and the product was partially dried under vacuum for 1.75 h. The product was removed from the funnel and placed in 6 glass plates. The trays were placed in a vacuum oven and the product was dried at 60 ℃ for 64.75h using a Nash pump as a vacuum source and a stream of argon. The pan was removed, weighed, placed back in the drying cabinet and dried at 60 ℃ for 4h to obtain a constant weight. The vacuum source for the second drying period was an oil pump, using no argon flow. The material was packaged in a double layer 4-mil polyethylene bag and placed in a plastic-shelled container. After sampling, the final weight was 2,540.9g (92.5%).

Example 2-

Effect of SAHA and Gemcitabine combination in T24 cell line

The combination of SAHA with gemcitabine resulted in the observation of a combined synergy that was greater than the additive effect obtained by the use of each drug alone.

Materials and methods：

The cells were aligned at 1.25X 10⁴Cells/ml density were seeded in MEM α medium containing 10% FCS and allowed to attach to the wells.

Gemcitabine was dissolved in MEM α medium and the pH was adjusted to 7 with 1N NaOH. Gemcitabine was prepared at each concentration by serial dilution of gemcitabine with complete medium. Each concentration of SAHA was prepared from a 1mM stock.

Cells were left untreated and treated with SAHA alone, gemcitabine alone or a combination of SAHA and gemcitabine simultaneously by aspiration of the wells and addition of the indicated concentrations of the relevant media. The cells are then cultured in a medium containing the compound or a combination of compounds.

To determine proliferation and viability, triplicate cell samples were harvested and proliferation and viability were counted at defined time points. To harvest the cells, the contents of each well were removed with 0.5ml trypsin, transferred to a 15ml tube, the cells centrifuged and resuspended in 1ml of medium. Harvested cells were counted on a hemocytometer for proliferation. Viability was determined by trypan blue exclusion.

Results：

T24 cells were cultured in complete medium (control) for 96 hours with 2nM gemcitabine, with 5 μ M SAHA or with a combination of 2nM gemcitabine and 5 μ M SAHA.

The results are plotted in fig. 1A (showing cell proliferation) and fig. 1B (showing cell viability). As shown in figure 1, cells treated with the combination of SAHA and gemcitabine inhibited significantly more cell proliferation than either SAHA or gemcitabine alone.

The combination of gemcitabine and SAHA produces significantly better results than the additive effect of the components when administered alone-i.e., a synergistic response, providing an increased advantage over the additive response.

Example 3-

Effect of SAHA and Gemcitabine combination in LnCap cell lines

Materials and methods：

The cells were aligned at 2.5X 10⁴Cells were plated at density/ml in RMPI medium with 10% FCS and allowed to attach to the wells.

Gemcitabine was dissolved in the medium and the pH was adjusted to 7 with 1N NaOH. Gemcitabine was prepared at each concentration by serial dilution of gemcitabine with complete medium. SAHA was prepared at each concentration from a 1mM stock solution.

To determine proliferation and viability, triplicate cell samples were harvested as described in example 2 above and proliferation and viability were counted at the indicated time points.

Results：

LnCap cells were cultured in complete medium (control) with 2nM gemcitabine, with 5 μ M SAHA or with a combination of 2nM gemcitabine and 5 μ M SAHA for 72 hours.

The results are plotted in fig. 2A (showing cell proliferation) and fig. 2B (showing cell viability). As shown in figure 2, treatment of cells with gemcitabine alone produced a small effect on cells, whereas treatment with SAHA significantly inhibited proliferation. Treatment with a combination of SAHA and gemcitabine produced additive effects.

Example 4-

Effect of SAHA and 5-azacytidine combination in T24 cell line

Materials and methods：

Cells were cultured in T-150 flasks at 37 ℃ with RPMI containing 10% FCS. Cells were diluted to 5.0X 10 with complete medium⁴Cell/ml density. Cells were incubated at 37 ℃ for 14 hours and then treated with 5-azacytidine to attach the cells to the wells.

5-azacytidine was prepared at each concentration by serial dilution from a 1mM stock solution. After 14 h incubation in complete medium, aspirate from the well and replace the medium with 1ml of 5-azacytidine at the indicated concentration. Cells were preincubated in 5-azacytidine for 27.5 hours, then SAHA was added.

SAHA was prepared at each concentration from a 1mM stock solution.

After preincubation in medium alone (control) or with 5-azacytidine, aspirate from the wells and replace the well contents with 1ml of medium alone (control), medium containing 5-azacytidine alone, medium containing SAHA alone, or medium containing a combination of 5-azacytidine and SAHA.

Results：

T24 cells were cultured in complete medium (control), 200nM 5-azacytidine, 5. mu.M SAHA or a combination of 200nM 5-azacytidine and 5. mu.MSHA as described above.

The results are plotted in fig. 3A (showing cell proliferation) and fig. 3B (showing cell viability). As shown in FIG. 3, treatment of cells with 5-azacytidine alone or SAHA alone significantly inhibited proliferation. Treatment with a combination of SAHA and 5-azacytidine produced an additive effect, resulting in substantially complete inhibition of proliferation relative to the initial cell count.

Example 5-

SAHA and Etoposide, Doxorubicin, 5-Fluorouracil, mitoxantrone and Oxasillid Use of combinations of platinum in breast, glioblastoma and prostate cancer cell lines

Purpose of study：

The purpose of these studies was to determine whether the combination of SAHA and the therapeutic agents listed in table 1 was more effective at inhibiting cell growth and colony formation than either drug alone. All combination drugs are commercially available and available from Sigma. In these studies, 5 different cell lines representing three general cancer types were tested (table 2). In both experiments, antiproliferative effects of several drugs were observed, and additive effects were generally observed.

TABLE 1 drugs for combination with SAHA

Therapeutic agents
Therapeutic agents	Etoposide (Eto)
Doxorubicin (Dox)	Etoposide (Eto)
Doxorubicin (Dox)	5-Fluorouracil (5-FU)
Mitoxantrone (Mitox)	5-Fluorouracil (5-FU)
Mitoxantrone (Mitox)	Oxaliplatin (Oxal)

TABLE 2 cell lines for SAHA combination studies

Cell lines
Cell lines	MDA-231 mammary gland
U-118 glioblastoma	MDA-231 mammary gland
U-118 glioblastoma	DU-145 prostate gland
PC-3 prostate gland	DU-145 prostate gland
PC-3 prostate gland	LnCap prostate

Cell growth assay：

The Cell growth inhibition Assay uses a commercially available MTS Assay, also known as the Cell Titer 96Aqueous One Solution Cell Proliferation Assay (96 well Cell Titer plate single Aqueous Solution Cell Proliferation Assay). MTS reagent containing [3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt, tetrazolium compound]And an electron coupling reagent (phenazine ethosulfate; PES). Assays were performed by adding small amounts of MTS reagent directly to culture wells, incubating for 1-4 hours, and then recording absorbance at 490nM using a 96-well plate reader. Formazan measured by absorbance at 490nMThe amount of product is directly proportional to the number of viable cells in the culture medium. The processing protocol for the MTS assay was performed in two different ways. In one method, the seeded cells were pretreated with SAHA for 4 hours, then washed to remove SAHA before the drug combination was added and the remainder incubated for 48 hours. In another method, cells are treated with SAHA for 48 hours, followed by addition of a second agent for 4 hours. Cells were then washed and allowed to grow for 48 hours.

Colony formation assay：

The colony formation assay was performed as follows. Cells were seeded at 200-300 cells/dish in 6cm dishes and allowed to attach for 24 hours. Cells were treated with SAHA for 48 hours, then the combination was added and held for an additional 4 hours. The cells were then washed and colonies were allowed to grow for 2-3 weeks and then stained with 2% crystal violet in methanol. All colonies with a threshold size (-0.2 mm) in each dish were counted. In each treatment group, dishes were counted in duplicate and the range of colony numbers/dish is shown as error bars.

Results：

A.SAHA combination pair MDA-231 Effect of cell proliferation (FIG. 4)

In one experiment, MDA-231 breast cancer cells were pretreated with the indicated concentration of SAHA for 4 hours, washed, and then the second drug was added and held for 48 hours. Cell growth was quantified using the MTS assay. The results are plotted in FIG. 4A.

In another experiment, cells were pretreated with the indicated concentration of SAHA for 48 hours, the second drug was added, held for 4 hours, and then the cells were washed. After 48 hours, cell growth was quantified using the MTS assay. The results are plotted in FIG. 4B.

As shown in figure 4, treatment with SAHA in combination with the indicated concentrations of the therapeutic drugs etoposide, doxorubicin, 5-fluorouracil, mitoxantrone and oxaliplatin produced a greater antiproliferative effect than treatment with each drug alone. The effect is clearly additive.

B.Effect of SAHA combination on DU145 cell proliferation (FIG. 5)

Cells were pretreated with SAHA at the indicated concentration for 48 hours, the second drug was added, held for 4 hours, and then cells were washed. After 48 hours, cell growth was quantified using the MTS assay.

As shown in figure 5, treatment with SAHA in combination with the indicated concentrations of the therapeutic drugs etoposide, doxorubicin, 5-fluorouracil, mitoxantrone and oxaliplatin produced a greater antiproliferative effect than treatment with each drug alone. The effect is clearly additive.

C.Effect of SAHA combination on the clonogenic development of DU145 cells (FIG. 6)

Cells were treated with SAHA for 48 hours, then the second drug was added, held for 4 hours, and then the cells were washed. After 2-3 weeks, colony formation was assessed.

As shown in figure 6, treatment with SAHA in combination with the indicated concentrations of the therapeutic drugs etoposide, doxorubicin and oxaliplatin reduced the number of colonies to a greater extent than treatment with each drug alone. The effect is clearly additive.

D.Effect of SAHA combination on MDA-231 cell clonogenesis(FIG. 7)

As shown in figure 7, treatment with SAHA in combination with the indicated concentrations of the therapeutic drugs etoposide, doxorubicin, 5-fluorouracil and oxaliplatin reduced colony numbers to a greater extent than treatment with each drug alone. The effect is clearly additive.

E.Effect of SAHA combination on U118 cell clonogeny(FIG. 8)

As shown in figure 8, treatment with SAHA in combination with the indicated concentrations of the therapeutic drugs etoposide, doxorubicin, 5-fluorouracil and oxaliplatin reduced colony numbers to a greater extent than treatment with each drug alone. The effect is clearly additive.

Example 6-

Combined effect of SAHA and chemotherapeutic agents irinotecan, 5-fluorouracil and docetaxel Purpose and summary of the study：

The aim of these studies was to administer three clinically used anticancer drugs in vitro: irinotecan, 5-fluorouracil (5-FU) and docetaxel in combination with SAHA, the effect of SAHA on transformed bladder cancer (T24), prostate cancer (LnCap), breast cancer (MCF7), non-hodgkin lymphoma (DLCL) and colon carcinoid tumor (LCC18) cell lines was evaluated.

Transformed cells were treated with various combinations of SAHA and one of these drugs in order to assess whether each pair (SAHA plus drug) produced additive, synergistic or antagonistic antiproliferative effects. The results indicate that the combined effect of SAHA with irinotecan, 5-fluorouracil and docetaxel is mostly additive. In some experiments, a synergistic effect was found. As described below, the most significant synergy occurred in LnCap cells with SAHA and docetaxel in combination.

Irinotecan, 5-fluorouracil and docetaxel

The three drugs are currently used in cancer chemotherapy in clinic and are all widely characterized.

Irinotecan functions to stabilize the nuclear topoisomerase I/DNA complex, which leads to accumulation of single strands of DNA breaks, leading to apoptosis. Clinically, it is used to treat a wide variety of cancers including breast, colorectal, cervical, ovarian and small/non-small cell lung cancers (Hardman W.E. et al (1999) Br J Cancer 81, 440-.

5-Fluorouracil (5-FU) acts as a pyrimidine antagonist, inhibits methylation of deoxyuridylate to thymidylate and subsequent synthesis of DNA and RNA(http://www.nursespdr.com/members/database/ndrhtml/fluorouracil.html). This drug has been widely used in recent decades as a chemotherapeutic drug, and has also been used clinically in combination with other anticancer drugs including irinotecan (Awada A. et al (2002) Eur J. cancer 38, 773-778).

Docetaxel is used clinically as an antitumor agent that disrupts the microtubule network in tumor cells, which can help inhibit cell division. By binding to free tubulin, it facilitates assembly and inhibits microtubule depolymerization (Chou T. et al (1991) synergy and Antagonism in chemotherapy. New York: Academic Press).

Materials and methods：

Drug combination experiments were performed in 5 human cancer cell lines: t24 (bladder cancer), LnCap (prostate), MCF7 (breast), DLCL (non-hodgkin lymphoma) and LCC18 (colon carcinoid tumor). Each cell line was cultured and incubated at 37 ℃ in the following media as required: MEM α (10% FCS), RPMI 1640 (10% FCS), DME HG (10% FCS), enhanced RPMI (10% FCS) and Hites medium (5% FCS).

Adherent cell lines (T24, LnCap, MCF7, and LCC18) were placed in 96-well plates for 24 hours and then treated with SAHA and anticancer drugs for a time to allow cells to attach to the bottom of the wells. On the same day of the experiment, DLCL cell line suspensions were placed in 96-well plates. For T24, LnCap, DLCL and LCC18, 200 μ L containing 2000 cells were seeded into each well. For MCF7, cells were seeded at 4000 cells/well for slow cell cycle counting of this cell line. In each SAHA and anticancer drug combination experiment, cells were seeded in two 96-well plates.

On the same day of treatment, all SAHA and combination drug treatments were prepared with cell line specific media. Cells that received no treatment served as a control group.

To administer treatment adherent cell lines (T24, LnCap, MCF7), the medium in each well was withdrawn and replaced with 200. mu.L of medium containing the desired concentration of drug or drugs. For all other cell lines (OCC18 and DLCL), 22. mu.L of medium containing 10-fold the desired concentration of drug or drugs was added to 200. mu.L of medium in each well.

After treatment, the cells were incubated at 37 ℃ for 4 days. On the fourth day, 20 μ LalamarBlur was used^TM(an aqueous dye) each well was treated and incubated at 37 ℃ for 4 hours. When adsorbed by cells, the reduction of the dye is greater in proliferating cells than in non-proliferating cells because the concentrations of NADPH, FADH, FMNH and NADH are increased. Using SpectaMaxSpectrofluorometer microtiter well plate reader (Molecular devices corporation, Sunnyvale, Calif.) the reduction was measured by fluorescence. Data are expressed in fluorescence emission intensity units as a function of incubation time with SOFTMaxv.4.0 software (Molecular Devices Corp. Sunnyvale, Calif.). To evaluate the percent inhibition of cell growth after 4 days of drug treatment, the following formula was used:

100- (mean intensity units/well with same treatment) 100 x 100

(average intensity units/well for control group)

The percent standard error for each percent inhibition evaluated was calculated using the following formula:

100-(intensity units/well standard error with same treatment) 100

(intensity units/well standard error of control group)

The treatment concentration of each drug used was based on the dose that inhibited 50% proliferation of each drug. The following concentrations were tested:

a) 50% effective dose/4; b) 50% effective dose/2; c) 50% effective dose; d) 50% effective dose x 2; and e) 50% effective dose 4. The dose at which each drug inhibited 50% proliferation was determined in preliminary experiments in which cells were treated with various concentrations of SAHA and irinotecan alone, 5-FU and docetaxel alone. A polynomial relationship was defined between drug concentration and percent inhibition with various concentrations, and this function was used to predict the concentration of each drug required to inhibit cell proliferation by 50%.

The following criteria were established for evaluating additive, synergistic and antagonistic interactions:

additive interactions will be observed if the percent cytostatic with the SAHA drug combination treatment is greater than the percent cytostatic with either SAHA alone or anticancer drug alone, but less than or equal to the percent cytostatic that would be expected if the drug combination treatment were purely additive (i.e., percent SAHA inhibition alone + percent drug inhibition alone).

Synergy will be observed if the percent cellular inhibition treated with the SAHA drug combination is greater than the percent cellular inhibition expected if the drug combination is treated purely additively (i.e., percent SAHA inhibition alone + percent drug inhibition alone).

Antagonistic interactions would be observed if the percent of cell inhibition with SAHA alone or anticancer drug alone was greater than the expected percent of cell inhibition with the drug combination.

Results：

A.Determination of effective dose for 50% cell proliferation：

50% cell proliferation effective dose of irinotecan, 5-FU and docetaxel in combination with increasing concentration of SAHA was analyzed. The results are shown in tables 3 and 4. The combination of SAHA and each anticancer drug resulted in a reduction in the drug concentration required to inhibit cell growth by 50%. In almost every experiment, increasing SAHA concentration resulted in lower drug concentrations required to achieve this inhibitory amount. For example, 5.1nM of irinotecan alone is required to inhibit 50% of T24 bladder cancer cells; however, when 0.625 μ M SAHA and irinotecan were administered in combination, the required drug concentration was reduced to 4.1 nM. For example, as the SAHA concentration further increases to 2.5 μ M, the required irinotecan concentration continues to decrease (1.9 nM).

Table 3: t2450% inhibition effective dose of representative combination treatment

	Alone	SAHA(0.625*)	SAHA(1.25)	SAHA(2.5)	SAHA(5.0)	SAHA(10.0)
	Alone	SAHA(0.625*)	SAHA(1.25)	SAHA(2.5)	SAHA(5.0)	SAHA(10.0)	Irinotecan (nM)	5.1	4.1	2.8	1.9	1.4	0.28
5-FU(μM)	14.0	13.1	11.5	--	2.7	1.5	Irinotecan (nM)	5.1	4.1	2.8	1.9	1.4	0.28
5-FU(μM)	14.0	13.1	11.5	--	2.7	1.5	Docetaxel (nM)	2.1	0.51	0.72	0.59	0.29	0.05

All SAHA concentrations were μ M. SAHA alone: 5.0. mu.M

Table 4: LnCap 50% inhibition, effective dose of representative combination treatment

	Alone	SAHA(0.125*)	SAHA(0.25)	SAHA(0.5)	SAHA(1.0)	SAHA(2.0)
	Alone	SAHA(0.125*)	SAHA(0.25)	SAHA(0.5)	SAHA(1.0)	SAHA(2.0)	Irinotecan (nM)	5.1	5.1	4.1	3.8	1.9	1.9
5-FU(μM)	3.0	5.2	--	1.6	1.9	0.99	Irinotecan (nM)	5.1	5.1	4.1	3.8	1.9	1.9
5-FU(μM)	3.0	5.2	--	1.6	1.9	0.99	Docetaxel (nM)	2.1	1.2	1.2	0.6	0.34	0.12

All SAHA concentrations were μ M. SAHA alone: 1.7. mu.M

B.SAHA and irinotecan：

SAHA and irinotecan combinations were tested in 4 cell lines: t24, LnCap, DLCL, and LCC 18. The interactions in all 4 cell lines were evaluated to be mostly additive.

Results are expressed as the percentage of each type of interaction (additive, synergistic and antagonistic) to the total effect (100%) of the combination treatment tested.

Table 5: observed in different cell lines after treatment with a combination of SAHA and irinotecan Interaction of

Cell type	Addition (%)	Synergy (%)	Antagonism (%)
Cell type	Addition (%)	Synergy (%)	Antagonism (%)	T24	86	0	14
LCC18	62	19	19	T24	86	0	14
LCC18	62	19	19	DLCL	50	10	40
LnCap	59	25	16	DLCL	50	10	40

In T24 bladder cancer cells, 86% of the combined treatments produced additive interactions. No synergy was reported. The 14% treatment produced antagonistic interactions. The additive interaction occurs in the treatment of high and low concentrations of SAHA and irinotecan (SAHA: 0.625-10 μ M, irinotecan: 2.6-10 nM).

In LnCap prostate cell line, 25% of combined treatments produced synergy. The 59% treatment produced additive interactions, most of which occurred in SAHA at high, neutralized concentrations and irinotecan from low to high concentrations (SAHA: 0.5-2. mu.M; irinotecan: 2.6-10 nM).

Combined treatment of DLCL resulted in half of the additive interaction, 40% being antagonistic. Antagonism mostly occurs at higher concentrations.

LCC18 cells at 62% treatment exhibited additive interaction, while 19% were synergistic interaction, and antagonism at the same percentage. SAHA in any concentration range is not specific for the interaction, but all synergistic interactions occur at low irinotecan concentrations (1.9 nM).

Representative curves representing individual responses are plotted in fig. 9 (LnCap). Similar curves were plotted for other cell lines (not shown).

B.SAHA and 5-Fluorouracil：

Most SAHA and 5-FU combination treatments produce additive interactions, as do irinotecan combinations. SAHA and 5-FU combinations were tested in 4 cell lines: t24, LnCap and LCC 18.

Table 6: observed in different cell lines after treatment with a combination of SAHA and 5-FU Interaction of

Cell type	Addition (%)	Synergy (%)	Antagonism (%)
Cell type	Addition (%)	Synergy (%)	Antagonism (%)	T24	80	6	14
LCC18	49	7	44	T24	80	6	14
LCC18	49	7	44	LnCap	60	33	7

In T24 bladder cancer cells, 80% of the combined treatments produced additive interactions. Most of these effects occur at high concentrations of SAHA (2.5. mu.M-10. mu.M).

In LnCap prostate cell line, 33% combined treatment resulted in synergy. The 60% treatment produced additive interactions, most of which occurred at high concentrations of SAHA (1.0-2.0. mu.M). Most of the synergy occurred at low SAHA concentrations (0.5. mu.M)

Treatment with 49% LCC18 produced additive interactions. Treatment with 44% LCC18 resulted in antagonism. The interaction does not occur within any particular concentration range.

Representative curves representing individual responses are plotted in fig. 10 (LnCap). Similar curves were plotted for other cell lines (not shown).

C.SAHA and docetaxel：

SAHA and docetaxel combination experiments were performed on T24, LnCap and LCC18 cells.

Table 7: after treatment with a combination of SAHA and docetaxel, observations in different cell lines Interaction of (2)

Cell type	Addition (%)	Synergy (%)	Antagonism (%)
Cell type	Addition (%)	Synergy (%)	Antagonism (%)	T24	80	5	15
LCC18	72	8	20	T24	80	5	15
LCC18	72	8	20	LnCap	38	56	6

In T24 bladder cancer cells, 80% of the combined treatments produced additive interactions, and 5% of the interactions were synergistic.

The greatest synergy (56%) occurred in experiments with SAHA/docetaxel combination in LnCap cells. This synergy occurs mainly in the low and medium SAHA concentration range (0.25-1. mu.M). The 38% treatment produced additive interactions at most high concentrations of SAHA (2.0 μ M).

LCC18 cells were treated in combination, resulting in 72% additive and 8% synergistic, respectively.

Representative curves representing individual responses are plotted in fig. 11 (LnCap). Similar curves were plotted for other cell lines (not shown).

Taken together, these results indicate that in general SAHA interacts mostly additively with irinotecan, 5-FU and docetaxel. However, it is important that the interaction of SAHA and docetaxel in LnCap cells is mostly synergistic. Other synergy was found at several concentrations tested, especially in LnCap cell lines.

Conclusion：

The results of all the above combination studies indicate that treatment with SAHA in combination with other anticancer drugs can be used for cancer therapy, since the dose of each drug in the combination therapy can be reduced compared to single drug therapy, while still obtaining the overall antitumor effect. Combination therapy is particularly useful when a synergistic effect between the two drugs is found as described above.

While the invention has been shown and described with reference to a preferred embodiment 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 is defined by the claims.

Claims

1.A first amount of suberoylanilide hydroxamic acid or a pharmaceutically acceptable salt or hydrate thereof represented by the structure:

and a second amount of docetaxel for use in the manufacture of a medicament for the treatment of cancer.

2. The pharmaceutical composition of claim 1, wherein the cancer is breast cancer.

3. The pharmaceutical composition of claim 1, wherein the cancer is prostate cancer.