NZ568678A

NZ568678A - Methods of inducing terminal differentiation using suberoylanilide hydroxamic acid (SAHA)

Info

Publication number: NZ568678A
Application number: NZ56867803A
Authority: NZ
Inventors: Victoria M Richon; Thomas A Miller; Judy Chiao; W Kevin Kelly
Original assignee: Merck Hdac Res Llc; Sloan Kettering Inst Cancer
Priority date: 2002-03-04
Filing date: 2003-03-04
Publication date: 2010-08-27

Abstract

Disclosed is a crystalline form of suberoylanilide hydroxamic acid (SAHA) obtained by recrystallizing a crude preparation of SHA from a mixture of methanol and water. Also disclosed is a crystalline form of SAHA obtained by reacting suberic acid with aniline to obtain suberanilic acid, reacting the suberanilic acid with methanol to form methyl suberanilate, reacting the methyl suberanilate with hydroxylamine hydrochloride to form crude SAHA, and recrystallizing the crude SAHA from a mixture of methanol and water. (62) Divided Out of 550185

Description

<div class="application article clearfix" id="description"> 568678 *10055925598* Se8678 28 MAY 2008 RECEIVED PATENTS FORM NO. 5 Our ref: WIB 507392NZPR Divisional of NZ 550185 which is in turn a Divisional of NZ 535578 Antedating requested to 4 March 2003 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION METHODS OF INDUCING TERMINAL DIFFERENTIATION We, SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH of 1275 York Avenue, New York, 10021, New York, United States of America and MERCK HDAC RESEARCH, LLC of 33 Avenue Louis Pasteur, Boston, 02115, Massachusetts, United States of America hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: -1 - The next page is numbered "1A" 300908731 _1 ,DOC:WIB:hew 568678 WO 63/4)7583$ PCT/US03A»645I METHODS Of INDUCING TERMINAL DIFFERENTIATION CROS^REFERBNCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 5 60/361,759, fled March 4, 2002. lie entire teachings of this provisional application are incorporated herein by reference. GOVERNMENT INTEREST STATEMENT This invention was made in whole or in part ■with government support under grant number LR21 CA 096228-01 awarded by the National Cancer Institute. The government I 10 may have certain rights in fee invention. The present invention provides methods of selectively inducing terminal differentiation, cell growth arrest arid/or apoptosis of neoplastic cells, and/or inhibiting IS histone deacetylases (HDAC) administration of pharmaceutical compositions comprising HDAC inhibitors. The oral formulations of the pharmaceutical compositions have favorable pharmacokinetic profiles such as high bioavailability and surprisingly give rise to high blood levels of the active compounds over an extended period of time. Throughout this application various publications are referenced by arabic numerals within parentheses. Full citations for these pubKcations may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in 25 fully describe fee state of the art to which this invention pertains. Cancel is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation. For many years there have been two principal strategies for chemotherapeutic treatment of cancer a) blocking hormone-dependent tumor cell 30 proliferation by interference with the production or peripheral action of sex hormones; and b) HTHtip cancer cells directly by exposing them to cytotoxic substances, which injure both Cancer therapy is also bang attempted by the induction of terminal differentiation of the neoplastic cells (1). ia cell culture models differentiation has been reported by FIELD OF THE INVENTION 20 BACKGROUND OF THE INVENTION their entireties are hereby incorporated by reference into this application in order to more 1A 568678 WO 03/075839 PCT/US03/06451 exposure of cells to a variety of stimuli, including: cyclic AMP and retmoic acid (2,3), aclanibicin and other anthracyclines (4). Despite many advances in fee field of oncology, the majority of solid tumors remain incurable in the advanced stages. Cytotoxic therapy is used in most cases, 5 however, it often causes significant morbidity to fee patient without significant clinical benefit. Less toxic and more specific agents to treat and control advanced malignancies are being explored. There is abundant evidence that neoplastic transformation does not necessarily destroy the potential of cancer cells to differentiate (1,5,6). There are many examples of 10 tumor cells which do not respond to the normal regulators of proliferation and appear to be blocked in the expression of their differentiation program, and yet can be induced to differentiate and cease replicating. A variety of agents, including some relatively simple polar compounds (5,7-9), derivatives of vitamin D and retinoic acid (10-12), steroid hormcecs (13), growth factors (6,14), proteases (15,16), tumor promoters (17,18), and 15 inhibitors of DNA or RNA synthesis (4,19-24), can induce various transformed cell lines and primary human tumor explants to express mare differentiated characteristics. Early studies identified a series of polar compounds that were effective inducers of differentiation in a number of transformed cell lines (8,9). Of these, the most effective inducer "was the hybrid polar/apolar compound N.N'-hexamethylene bisacetamide 20 (HMBA) (9). The use of this polar/apolar compound to induce murine erythroleukemia cells (MELC) to undergo erythroid differentiation with suppression of oncogenicity has proved a useful model to study inducer-mediated differentiation of transformed cells (5,7-9). HMBA-induced MELC terminal erythroid differentiation is a multi-step process. Upon addition of HMBA to MELC (745A-DS19) in culture, there is a latent period of 10 to 12 25 hours before commitment to tenninal differentiation is detected. Commitment is defined as the capacity of cells to express tenninal differentiation despite removal of inducer (25). Upon continued exposure to HMBA there is progressive recruitment of cells to differentiate. The present inventors have reported that MELC cell lines made resistant to relatively low levels of vincristine become markedly more sensitive to the inducing action 30 of HMBA and can be induced to differentiate with little or no latent period (26). HMBA is capable of inducing phenotypic changes consistent with differentiation in a broad variety of cells lines (5). The characteristics of the drug-induced effect have been most extensively studied in the murine erythroleukemia cell system (MELC) (5,25,27,28). MELC induction of differentiation is both time and concentration dependent 568678 WO 03/075839 PCT/US03/06451 The mininram conceoiratioB required to demonstrate an effect in vitro in most strains is 2 to 3 xnM; the rnfnTmirm duration of continuous exposure generally required to induce differentiation, in a substantial portion (> 20%) of the population without continuing drug exposure is about 36 hours. 5 The primary target of action of HMBA is not known. There is evidence that protein kinase C is involved in the pathway of inducer-meaiated differentiation (29). The in vitro studies provided a basis for evaluating the potential of HMBA as a cytodifferentiaiion agent in fee treatment of human cancers (30). Several phase I clinical trials wife HMBA have been completed (31-36). Clinical trials have shown that this compound can induce a 10 therapeutic response in patients -wife cancer (35,36). However, these phase I clinical trials also have demonstrated feat fee potential efficacy of HMBA is limited, in part, by dose-related toxicity which prevents achieving optimal blood levels and by fee need for intravenous administration of large quantities of the agent, over prolonged periods. It has been reported feat a number of compounds related to HMBA wife polar 15 groups separated by apolar linkages feat, on a molar basis, are as active (37) or 100 times more active than HMBA (38). As a class, however, it has been found feat the symmetrical . (timers such as HMBA and related compounds are not fee best cytodifferentiatiiig agents. It has unexpectedly been found that fee best compounds comprise two polar end groups separated by a flexible chain of methylene groups, wherein one or bofe of fee polar -20 end groups is a large hydrophobic group. Preferably, the polar end groups are different and only one is a large hydrophobic group. These compounds are unexpectedly a thousand times more active than HMBA and ten times more active than HMBA related compounds. Histone deacetylase inhibitors such as suberoylsmlide hydroxamide acid (SAHA), belong to this class of agents feat have the ability to induce tumor cell growth arrest, 25 differentiation and/or apoptosis (39). These compounds are targeted towards mechanisms inherent to fee ability of a neoplastic cell to become malignant, as they do not appear to have toxicity in doses effective for inhibition of tumor growth in animals (40). There are several lines of evidence feat histone acetylation and deacetylation are mechanisms by which transcriptional regulation in a cell is achieved (41). These effects are thought to 30 occur through changes in fee structure of chromatin by altering fee affinity of histone proteins for coiled DNA in the nucleosome. There are five types of histones that have been identified in nucleosomes (designated HI, H2A, H2B, H3 and H4). Each nucleosome contains two of each histone type within its core, except for HI, which is present singly in fee outer portion of fee nucleosome structure. It is believed feat when fee 568678 WO 03/075839 PCT/CS03/0M51 histone proteins are hypoacetylated, there is a greater affinity of the histone to the DNA phosphate backbone This affinity causes DNA to be tightly bound to the histone and readers the DNA inaccessible to transcriptional regulatory elements and machinery. The regulation of acetylated states occurs through the balance of activity between two enzyme 5 complexes, histone acetyl transferase (HAT) and histone deacetylase (HDAC). The hypoacetylated state is thought to inhibit transcription of associated DNA. This hypoacetylated state is catalyzed by large multiprotein complexes that include HDAC enzymes, hi particular, BDACs have been shown to catalyze the removal of acetyl groups from the chromatin core histones. 10 The inhibition of HDAC by SAHA is thought occur through direct interaction with the catalytic site of the enzyme as demonstrated by X-ray crystallography studies (42). The result of HDAC inhibition is not believed to have a generalized effect on the genome, but rather, only affects a small subset of the genome (43). Evidence provided by DNA microarrays using malignant cell lines cultured with a HDAC inhibitor shows that there 15 are a finite (1-2%) number of genes whose products are altered. For example, cells treated in culture with HDAC inhibitors show a consistent induction of the cyclin-dependerrt kinase inhibitor p21 (44). This protein plays an important role in cell cycle arrest. HDAC inhibitors are thought to increase the rate of transcription of p21 by propagating the hyperacetylated state of histones in the region of the p21 gene, thereby malHng the gene 20 accessible to transcriptional machinery. Genes whose expression is not affected by HDAC inhibitors do not display changes in the acetylation of regional associated histones (45). It has been shown in several instances that fee disruption of HAT or HDAC activity is implicated in the development of a malignant phenotype. For instance, in acute promyelocytic leukemia, the oncoprotein produced by the fusion. of PML and RAR alpha 25 appears to suppress specific gene transcription through the recruitment of HDACs (46). In this manner, the neoplastic cell is unable to complete differentiation and leads to excess proliferation of the leukemic cell line. U.S. Patent Numbeis 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 30 inducing terminal differentiation of neoplastic cells, which compounds have two polar end groups separated by a flexible chain of methylene groups or a by a rigid phenyl group, wherein one or both of the polar end groups is a large hydrophobic group. Some of the compounds have an additional large hydrophobic group at the same and of the molecule as the first hydrophobic group which further increases differentiation activity about 100 fold WO 03/C75J3 568678 PCT7IJSC3/D6451 in se snrymaJie assay and &aat 50 sold in a «£ difBrsoiia&an. assay, Methods of syafbesismg fee canmoands used in Sic methods and ptharmacsorical GnTTroosrijons of inis mvsnaoo are fully dsscrfbsd foe aforementioned pssaEts, iiie satire contsnts of VKfakh are maxwjmtad harem "by rsfsraaes. 5 lbs- afbrsassmaaed patsoiE do not disclose specific csral fbrmctetiaas of fee HDAC mVtiVroTTra ar specific dosages and dosing sdhednlss of the resrtsd compounds. Tmprgtanfty fiis assrsmmtioned patents do not discloss oral fcantoMaas feat have favorable pharmacokinetic pro-files sock as Mai bioavailability winch gives rise to Mgii "blood levels of fee active campotrnds over an errmdsd period of time. 10 The class of compounds of fee present inrsniian may be oseSil for selectively fnfi-infr>o- terminal diffH^soiiatiaQ of oeoplastic cells and therefore aid in treatrasEt of tnmors id patients. Tims there is an urgent ncsd to discover saitaible dosages and dosing schedules of these compounds. sad to develop fommlafiaEis, preferably oral fonmilaiions, ■which give rise to steady, tfaerapesntically effective blood levels of fee active compounds 15 over an extended period of time, or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION The present invention provides methods of producing a mean plasma concentration of a hastcme deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in 20 vrvo in a subject over a period of at least two honrs following administration, which comprises administering to said subject an effective amount of a. pharmaceutical composition comprising a HDAC inhibitor or a phsrm sceptically acceptable salt or hydrate thereof and a pharmacenficaUy acceptable earner or ifr'Tiignt The present invention also provides methods for selectively inducing tenninal 25 difEsreafiatioii, cell growth arrest .and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of a histone deacetylase (BDAC) inhibitor enable of ^rniffr.0- a histone deacetylase in vrvo in a snbject over a period of at least two hoars following administration, by administering to said snbject an effective amonnt of a 30 phanoaceatical composition comprising a HDAC inhibitor or a pharmacenticElly acceptable salt or hydiate thereof. and a phannacectically acceptable cams or fflnwrf The present iuvtaiiian firrfher provides me&ods of producing a mean plasma concentration of st least about 10 nM of sribesoylamlide bydroxamic add (SABA) in vrvc in a subject ovar a pesiod of at least two hours following administration, which, comprises i 568678 WO G3/C75E39 PCT/USS/W451 ai4nnmig>"'^rTTio to said sabjsct an effective srtcsnnr of a pfcansacasiiGal caDixpositian ynrnp-igrnc SAHA ar a phaiiDacsaiically ass^rtabls sait ar bydrsts 'flisrao^ and a pkaosacsntieally acceptable earns ar dDnait Ths prsssst inreaacE also provides msfiiods for selestiveiy inducing tenninal 5 ifigrinn^ csfl grovrfk Brest aad/oi spoptosis of asqplasiic cells. thereby inhjbinag proliferatioiL of sndi cdk, aad meffaods far frirforrng diSramation of tamor cells "by producing a mean plasma eancsaiirafciaa of at least 10 dM of SAEA in vivo in a subject ova: a period of at least two krars following adnmrisbstirai, by artrnmistermg to said subject an effective amonnl of a pitanaacsniical compositioa comprising SAHA or a 10 phannacsoiically acceptable salt or hydrate thereof and a phannaceiticaJly acceptable earner or diiaeaL The present invention provides pharmaceutical compositions suitable for oral admimstration, wfeicih edmprise a compound' useful for selectively inducing 1-rrmfnal rff ■frkrrrifi aiinnj cell guwih arrest and/or apoptosis of neoplastic cells, Bnd.hr winch is a 15 potent 4n~hihifrrr of histone deacetylase (HDAC). The phannaceatical conipositions are firmer comprised of mjcrocrysMTHne cellulose, croscannellose sodium and magnesium ph-arafe, The present invention also provides for pharmaceutical compositions for oral administration, comprising SAHA, microcrystaffine cellulose, croscannellose sodium and mapnPCTim stearale. Tie oral bioaYaila&ility of the active comporaids in the fbnnnlafions 20 of Ifae present invention, is surprisingly MgL Moreover, the formulations unexpectedly give rise to high, therapeutically effective blood levels of the active compounds over an extended period of time. The present invention further provides" a safe, daily rinsing regimen of these fonimlations, -which is easy to follow and to adhere to. The invention, further provides the use of SAHA or a pharmaceuticaliy 25 acceptable salt or hydrate thereof in the preparation of medicaments for the treatment of non-Hodgkins lymphoma, myelodysplastic syndrome, leukemia, cancer, and/or cancers with leukocyte infiltratioa The invention further provides kits comprising at least one pharmaceuticaliy effective unit dosage of SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof and instructions for the treatment of non-Hodgkins lymphoma, myelodysplastic syndrome, leukemia and/or cancers having leukocyte infiltration. 5 7P0 E»rtr?5E3S 568678 PCT/USC3/&W£i As dsEODsistraEEi aarssn, it b2£ bssn sanjosngiy aad tntsspsssdly £naad fhsi arsl .gsazaiffiicsiB caasnsmg HDAC sMbifors, pamealady snbsrojianffids bydrosasac.. add (SAHA), bays 'very high overall oral bhsysDsbDity o-f me asiive at;,riijjqai in vfvs. Hnrfriw. mi i, fhs fmnsilaaonB hits rise to ingh biood icvsk of fat accTe KEnpanad, vftnrh XBBsta nrtspestediy high over an sctanded psdod erf tint®, iar szsspit, igj to 10-12 boors. Ths oral fammkiioELH of fije prsssnt kvsmioii bare many advantages, sspscaaDy wbsn compared to parsnEeral fonrtnlaikEis, since can the cxae sod they provide hig^ siaile sad prolonged faswjeaEiealiy E^scm^ bbod levels of ED AC mhibitm.. and on &c omer bad are easy to adnnnistsr to pstiarfe "by any aogvsnncioril laads of m-sl admisistratiQii 6A 568678 WO 03/075839 PCT/US03/06451 Accordingly, the present invention provides a pjbarmaeeutical composition for oral administration comprising a histone deacetylase (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof, and a phannacetmcally acceptable carrier or diluent; wherein foe composition provides a mean plasma concentration of the HDAC inhibitor 5 effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 10 hours following administration. In a preferred embodiment, the present invention provides a pharmaceutical 10 composition for oral administration comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof; and a phanxtaceutically acceptable easier or diluent; wherein tie composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a 15 period of at least 10 hours following administration. The formulations of the present invention are useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells and therefore aid in treatment of tumors in patients. Accordingly, the present invention also provides a method of selectively inducing 20 tenninal differentiation of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof; and a pharmaceuticaliy acceptable carrier or diluent; wherein the composition provides a mean 25 plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. Furthermore, the present invention also provides a method of selectively inducing cell growth arrest of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising the step of orally administering to the subject an 30 effective amount of a pharmaceutical composition comprising -a histone deacetylase (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or effluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. 568678 WO 03/075E39 PCT/SSfO/OMSl Furthermore, the present invsnfion also provides a method of selectively inducing apqptoBis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells m the subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) 5 inhibitor or a pharmacsnticaHy acceptable salt or hydrate thereof; aad a phannaceoticaSy acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. Furthermore, the present invention also provides a mefhod of inducing 10 differentiation of tumor ceils in a. subject having a tumor, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to 15 inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase in a subject, comprising the step of orally administering to the subject an effective amount of a pharmaceutical composition comprising a histone deacetylase 20 (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof; and a pharmaceuticaliy acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. Furthermore, the present invention also provides a method of selectively inducing 25 terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in a subject aad thereby inhibiting proliferation of such cells in the subject, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone 30 deacetylase (HDAC) m vivo for a period of at least 2 hours following administration. Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tninor comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising SAHA. or a pharmaceuticaliy acceptable salt or hydrate thereof; wherein the WO G3/C75S39 568678 PCT/TTSOS/OfrJSl composition provides a mean plasma concentration of SAHA effective to inhibit a. histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. Furthermore, the present invention provides a method of inhibiting 2k activity of a histone deacetylase in a subject, comprising ths step of orally administering to fee subject 5 an effective amount of a pharmaceutical composition comprising SAHA or a phannaceaiicaDy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of SAEA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of ai least 2 hours following administration. 10 In a preferred embodiment, SAHA or any of the HDAC inhibitors are administered to foe patient at a total daily dosage of between 25-4000 mg/m1. In another preferred embodiment, SAHA or any of the HDAC inhibitors are administered to the patient at a total daily dosage of 200 mg. SAHA or any of the HDAC inhibitors are administered to the patient at a total daily dosage of 400 mg. 15 M one preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) capable of inhibiting histone deacetylase over a period of at least 2 hours following administration, winch is preferably at a concentration, of at least aborit 10 nM. In yet another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor of at least 20 about 10 nM over a period of at least 10 horns following administration. in one preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor" (e.g., SAHA) capable of selectively inducing fen-ninai differentiation, cell growth arrest and/or apoptosis of neoplastic cells, or capable of inducing differentiation of tumor cells in a tumor, wherein the concentration is 25 maintained ova: a period of at least 2 hours following administration, which is preferably at a concentration of at least about 2.5 jjM. in yet another preferred embodiment, the composition provides a mean plasma, concentration of the HDAC inhibitor of at least about 2.5 ^Mover a period of at least 10 hours following administration. The compositions of the present invention may he formulated in any unit dosage 30 form (liquid or solid) suited for oral administration, for example, in the form of a pellet, a tablet, a coated tablet, a capsule, a gelatin capsule, a solution, a suspension, or a. dispersion, hi a preferred embodiment, the composition is in fee form of a gelatin capsule. 9 568678 WO 03/075839 PCT/US03/06451 Any inert sxcipient that is commonly used as a earner or diluent may be used in the formulations of the pressnt invention, such as for example, a gam, a stardi, a sugar, a cellnlosic material, an acryla±s: 01 mixtures thereof. A prefaned diluent is microcrystalline cellulose, Ths compositions may further comprise a disintegrating agent 5 (e.g., sodium croscanaeUose) and a lubricant (e.g.s magnesium stearate), and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a ■viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof Furthermore, the compositions of the present invention may be in the form of controlled 10 release or immediate release formulations. A wide variety of HDAC inhibitors are suitable for use in the compositions of the present invention, hi a preferred embodiment, fhe HDAC inhibitor is suberoylanilide hydxoxamic acid (SAHA). o NHOH 15 Other non-limiting examples of HDAC inhibitors that are suitable for use in fhe compositions of the present invention are: Pyroxamide, represented by the structure: o NHOH 20 A compound represented by the structure: 25 10 568678 WO 03/075839 PCT/US83/06451 wherein R3 and R4 are independently a substituted or unsubstituied, branched or uubraached alkyi, alkeayl, cycloalijd, aiyl, alkyfoxy, arylozy, arylalkyioxy, or pyridine group, cycloaBcyL aryi, aryioxy, arylalkylory, or pyridine group, or R3 and R4 bond together to form a piperidine group; S.2 is a 5 hydroxylamino group; and u is an integer from 5 to about 8. A compound represented by the structure: o 0 ■ v_J R C MH (CH^n C NHOH •wherein R is a substituted or nnsubstitated phenyl, piperidine, thiazole, 2-10 pyridine, 3- pyridine or 4-pyridine and e is an integer from about 4 to about 8. A compound represented by the structure: 0 Ris (Cl-yn .NHOH ,^n2jn y 0<* Y o wherein A is an amide moiety, Ri and R2 are each selected from substituted 15 or unsabstitoted aryl (e.g., phenyl), arylaUcyl (e.g., benzyl), naphthyl, pyridineanuno, 9-purine-S-arnino, thiazoleamino, aryioxy, arylalkyloxy, pyridyl, qumolinyl or isoquinoEbyl; R» is hydrogen, a halogen, a phenyl or a cycloaBcyl moiety and n is an integer from 3 to 10. 20 Furthermore, in accordance with specific embodiments of .the present invention, there is provided a pharmaceutical composition for oral administration comprising a histone deacetylase (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof; microcrystalline cellulose as a carrier or diluent; croscarmellose sodium as a diantegrant; and magnesium stearate as a lubricant; ■wherein the composition provides a 25 mean plasma concentration of ths HDAC inhibitor effective to inhibit a histone deacetylase in vivo far a period of at least 2 hours following administration. 61 a preferred embodiment, the HDAC inhibitor is suberoylanihde hydiaxarrdc add (SAHA). Furthermore, in accordance with specific embodiments of fhe present invention, there is provided a pharmaceutical composition for oral administration comprising 11 568678 WO 03/075839 PCT/US03/06451 suberoylanilide hydroxamic acid (SAHA) or a pharmaceuticaliy acceptable salt or hydrate thereof; microcrystalline cellulose as a carrier or diluent; croscarmellose sodium as a disintegrant; and magnesium stearate as a lubricant; wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inihibit a histone deacetylase in vivo for 5 a period of at least 2 hours following administration. In another preferred embodiment, the composition comprises 50-70% by weight of SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof, 20-40% by weight microcrystalline cellulose as a carrier or diluent; 5-15% by weight croscarmellose sodium as a disintegrant; and 0.1-5% by weight magnesium stearate as a lubricant. In another preferred embodiment, the composition comprises about 50-200 mg of 10 SAHA. In a particularly preferred embodiment, the composition is in the form of a gelatin capsule. The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow and to adhere to. The formulations of the present invention 15 are useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells and therefore aid in treatment of tumors in patients. The invention further provides an active ingredient consisting of a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of SAHA obtainable by 20 a method comprising the step of recrystallizing a crude preparation of SAHA from a mixture of methanol and water. The invention further provides an active ingredient consisting of a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of SAHA obtained by a 25 method comprising the steps of: a. reacting suberic acid with aniline to form suberanilic acid having the structure: h O OH O or a salt thereof; 301227341 l.DOC 12 intellectual property OFFIOF or ,\|2 -4 SEP 2009 fi-r .ar- 568678 WO 03/075839 PCT/US03/06451 b. reacting the suberanilic acid or the salt thereof with methanol to form methyl suberanilate having the structure: c. reacting the methyl suberanilate with hydroxylamine hydrochloride to form a crude 5 suberoylanilide hydroxamic acid in a reaction mixture; and d. recrystailizing a crude preparation of SAHA from a mixture of methanol and water. The invention further provides a pharmaceutical composition comprising the active ingredient as defined previously and a pharmaceuticaliy acceptable carrier, wherein the 10 composition is formulated as a solid dosage form. The invention further provides a pharmaceutical composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtainable by a method comprising the step of recrystailizing a crude preparation of SAHA from a 15 mixture of methanol and water, and a pharmaceuticaliy acceptable carrier, wherein the composition is formulated as a solid dosage form. The invention further provides a pharmaceutical composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline 20 SAHA obtained by a method comprising the steps of: H r\ a. reacting suberic acid with aniline to form suberanilic acid having the structure: oh or a salt thereof; 301227341 l.DOC INTELLECTUAL PROPERTY OFFICE OF N.Z. 12A SEP 2009 RECEIVED 568678 WO 03/075839 PCT/US03/06451 b. reacting the suberanilic acid or the salt thereof with methanol to form methyl suberanilate having the structure: h O o 5 c. reacting the methyl suberanilate with hydroxylamine hydrochloride to form a crude suberoylanilide hydroxamic acid in a reaction mixture; and d. recrystailizing a crude preparation of SAHA from a mixture of methanol and water; and a pharmaceuticaliy acceptable carrier, 10 wherein the composition is formulated as a solid dosage form. The invention further provides a composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtainable by a method comprising the step of recrystailizing a crude preparation of SAHA from 15 a mixture of methanol and water. The invention further provides a composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtained by a method comprising the steps of: 20 a. reacting suberic acid with aniline to form suberanilic acid having the structure: H r\ OH or a salt thereof; b. reacting the suberanilic acid or the salt thereof with methanol to form methyl 25 suberanilate having the structure: 301227341 l.DOC 12B INTELLECTUAL PROPERTY office of n.z SEP 2009 RECEIVED 568678 300909336 1.DOC c. reacting the methyl suberanilate with hydroxylamine hydrochloride to form a crude suberoylanilide hydroxamic acid in a reaction mixture; and d. recrystailizing a crude preparation of SAHA from a mixture of methanol and water. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, features and advantages of the invention will be 10 apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. 15 FIG. 1 is a picture of a Western blot (top panel) showing the quantities of acetylated histone-4 (a-AcH4) in blood plasmas of patients following an oral or intravenous (IV) dose of SAHA. IV SAHA was administered at 200 mg infused over two hours. Oral SAHA was administered in a single capsule at 200 mg. The amount of a-AcH4 is shown at the indicated time 20 points. Bottom panel: Coomassie blue strain. FIG. 2 is a picture of a Western blot (top panels) showing the quantities of acetylated histone-4 (a-AcH4) in the blood plasma of patients having a solid tumor, following an oral or intravenous (IV) dose of SAHA. IV and 25 Oral SAHA were administered as in Figure 1. The amount of a-AcH4 is 12C WO 03/075839 568678 PCTYUS03/06451 shown at the indicated time points. The experiment is shown in duplicate (Fig 2A and Fig 2B). Bottom panels: Coomassie blue stain. PIG. 3 is a picture of a Western blot (top panels) showing the quantities of acetylated histone-4 (a-AcH4) (Figure 3A) and acetylated histone-3 (a-5 AcH3) (Figures 3B-E) in the blood plasma of patients following an oral or intravenous (IV) dose of SAHA, on Day 1 and Day 21. IV and Oral SAHA were administered as in Figure 1. Hie amount of a-AcH4 or a-AcH3 is shown at the indicated time points. Bottom panels: Coomassie blue stain. FIG. 4 is a picture of a Westem blot (top panels) showing the quantities of 10 acetylated Mstone-3 (a-AcH3) in the blood plasma of patients having a solid tumor, following an oral or intravenous (TV) dose of SAHA. IV and Oral SAHA were administered as in Figure 1. The amount of a-AcH3 is shown at the indicated time points. Bottom panel: Coomassie blue stain. HG. 5 is a picture of a Western blot (top panels) showing the quantities of 15 acetylated histone-3 (a-AcH3) in the blood plasma of patients following an oral or intravenous (IV) dose of SAHA. IV SAHA was administered at 400 pig infused over two hours. Oral SAHA was administered in a single capsule at 400 mg. The amount of a-AcH4 is shown at the indicated time points. The experiment is shown in triplicate (Fig 5A and B). Bottom 20 panels: Coomassie blue stain. TIG. 6 is a pictnre of a Westem blot (top pane!) showing the quantities of acetylated histone-3 (a-AcH3) in the blood plasma of patients having a solid tumor, following an oral or intravenous (IV) dose of SAHA. IV and Oral SAHA were administered as in Figure 5. The amount of ct-AcH3 is 25 shown at the indicated time points. Bottom panel: Coomassie blue stain. FIG. 7 is a picture of a Westem blot (top panels) showing the quantities of acetylated histone-3 (a-AcH3) in the blood plasma of patients having a solid tumor following an oral or intravenous (IV) dose of SAHA, on Day i and Day 21. IV and Oral SAHA were administered as in Figure 4. The 30 amount of a-AcH4 or a-AcH3 is shown at the indicated time points. The experiment is shown in triplicate (Fig 7 A-Q. Bottom panels: Coomassie blue stain. ITG. 8 is a picture of a Western blot (top panels) showing fhe quantities of 13 WO 03/075839 568678 PCT/US03/06451 acetylated histone-3 (a-AcH3) in the blood plasma of patients following an oral or intravenous (IV) dose of SAHA. IV aad Oral SAHA were administered as in Figure 5. The amount of a-AcH3 is shown at the indicated time points. Bottom panels: Coomassie blue stain. FIG. 9A-C are graphs showing the mean plasma concentration of SAHA (ng/ml) at the indicated time points following administration. Fig 9A: Oral dose (200 mg and 400 mg) under fasting on Day 8. Fig 9B: Oral dose with food on Day 9. Fig 9C: IV dose on day 1. FIG. 10 shows the apparent half-life of a SAHA 200 mg and 400 mg oral dose, on Days 8,9 and 22. FIG. 11 shows the AUC (ng/ml/hr) of a SAHA 200 mg and 400 mg oral dose, on Days 8, 9 and 22. FIG. 12 shows the bioavailability of SAHA after a 200 mg and 400 mg oral dose, on Days 8, 9 and 22. DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods of producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, which 20 comprises administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceuticaliy' acceptable salt or hydrate thereof, and a phaimaceuticalLy acceptable carrier or diluent The present invention also provides methods for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting 25 porliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of a histone deacetylase (HDAC) inhibitox capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration, by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceuticaliy 30 acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or diluent. The present invention further provides methods of producing a mean plasma concentration of at least about 10 nM of suberoylanilide hydroxamic acid (SAHA) in vivo in a subject over a period of at least two hours following administration, which comprises 10 14 WO 03/075839 568678 PCT/US03/06451 administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or diluent. The present invention also provides methods for selectively inducing terminal 5 differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, and methods for inducing differentiation of tumor cells by producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration, by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a 10 pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or diluent. The present invention provides pharmaceutical compositions suitable for oral administration, which comprise a compound useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and/or which is a 15 potent inhibitor of histone deacetylase (HDAC). The pharmaceutical compositions are further comprised of microcrystalline cellulose, croscarmellose sodium and magnesium stearate. The present invention also provides for pharmaceutical compositions for oral administration comprising SAHA, microcrystalline cellulose, croscarmellose sodium and magnesium stearate. The oral bioavailability of the active compounds in the formulations 20 of the present invention is surprisingly high. Moreover, the formulations "unexpectedly give rise to high, therapeutically effective blood levels of the active compounds over an extended period of time. The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow and to adhere to. The oral bioavailability of the active compounds in the formulations of the present 25 invention is surprisingly high. Moreover, the formulations unexpectedly give rise to high, therapeutically effective blood levels of the active compounds over an extended period of time. The present invention further provides a safe, daily dosing regimen of these formulations, which is easy to follow, and which gives rise to a therapeutically effective amount of the recited compounds in vivo. The formulations of the present invention are 30 useful for selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, and therefore aid in treatment of tumors in patients. As demonstrated herein, the pharmaceutical compositions provided in the present invention give rise to an initial mean plasma concentration (i.e., the concentration that is obtained immediately after administration of the formulation), which remains 15 WO 03/075839 568678 PCT7US03/06451 unexpectedly high over an extended period of time. As compared with parenteral formulations (such as IV formnlatiorts) having the same dosage, in which the active compounds clear almost immediately, the oral compositions retain a high mean plasma concentration of the active compound over an extended period of time, for at least 2 hours, 5 but more typically at least, 10 or 12 hours. Typically, the mean plasma concentration of the oral dosage formulations, does not drop below 50% of the initial mean plasma concentration for a period of time of up to 12 hours or even longer. Up until the findings of the present invention, intravenous administration of the HDAC inhibitors described herein has proven to be the most effective. The intravenous 10 administration of the compound must be performed continuously, i.e., daily, for a prolonged period of time, such as for at least 3 days and preferably more than 5 days. This obviously provides a heavy burden on the patient receiving this treatment. The unexpected and surprising findings of the present invention make it possible to formulate oral dosage forms that give rise to high and steady levels of the active compounds in-vivo, 15 without the need to continuously administer the drugs, by IV infusions, which provides a tremendous advantage for the patient receiving the treatment. Accordingly, the present invention provides a pharmaceutical composition for oral administration comprising a histone deacetylase (HDAC) inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or diluent; 20 wherein the composition provides a mean plasma concentration of the HDAC inhibitor effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 8 hours following administration. In another preferred embodiment, the concentration of the HDAC 25 inhibitor is effective to inhibit the HDAC for a period of at least 10 hours following administration. In another preferred embodiment, the concentration of the HDAC inhibitor is effective to inhibit the HDAC for a period of at least 12 hours following administration. In a preferred embodiment, the present invention provides a pharmaceutical composition for oral administration comprising SAHA or a pharmaceuticaliy acceptable 30 salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or diluent; wherein the composition provides a mean plasma concentration of SAHA effective to inhibit a histone deacetylase (HDAC) in vivo for a period of at least 2 hours following administration. In a preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a period of at least 8 hours following administration. In another preferred embodiment, the 16 WO 03/075839 568678 PCT7TTS03/06451 concentration of SAHA is effective to inhibit the HDAC for a period of at least 10 hours following administration. In another preferred embodiment, the concentration of SAHA is effective to inhibit the HDAC for a period of at least 12 hours following administration. The formulations of the present invention are useful for selectively inducing 5 terminal differentiation, cell growth airest and/or apoptosis of neoplastic cells and therefore aid in treatment of tumors in patients. Accordingly, the present invention also provides a method of selectively inducing terminal differentiation of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of a 10 HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof, and a phannaceutically acceptable earner or diluent. 15 Furthermore, the present invention also provides a method of selectively inducing cell growth arrest of neoplastic cells in a subject and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective 20 amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or diluent. Furthermore, the present invention also provides a method of selectively inducing apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation of such cells 25 in the subject, comprising producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or diluent 30 Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor, producing a mean plasma concentration of a HDAC inhibitor capable of inhibiting a histpne deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC 17 WO 03/075839 568678 PCF/DSC3/06451 inhibitor or a. pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or diluent. Furthermore, the present invention provides a method of inhibiting the activity of a histone deacetylase is a subject, comprising producing a mem plasma concentration of a 5 HDAC inhibitor capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising a HDAC inhibitor or a pharmaceuticaliy acceptable sah or hydrate thereof and a pharmaceuticaliy acceptable carrier or diluent 10 Fuitheimore, the present invention also provides a method 'of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in a subject and thereby inhibiting proliferation, of such cells in the subject, comprising producing a mean plasma concentration of SAHA capable of inhibiting a histone deacetylase in vrvo in a subject over a period of at least two hours following administration by administering 15 to sairl subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceutical acceptable ■ carrier or diluent. Furthermore, the present invention also provides a mefhod of selectively inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells in a subject 20 and thereby inhibiting proliferation of such cells in the subject, comprising producing a mean plasma concentration of at least 10 nil of SAHA in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or diluent 25 Furthermore, the present invention also provides a method of inducing differentiation of tumor cells in a subject having a tumor comprising producing a mean plasma concentration of SAHA capable of inhibiting a histone deacetylase in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a 30 pharmaceuticaliy acceptable salt or hydrate thereof) and a pharmaceuticaliy acceptable carrier or diluent Furthermore, the present invention also provides a method of inducing differentiation of tumor cdls in a subject having a tumor comprising producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at 18 WO 03/075839 568678 PCT/US03/06452 least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA ar a phannaceufcically acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable earner or rtTlnwnt Furthermore, the present invention provides a method of inhibiting the activity of a 5 histone deacetylase in a subject, comprising producing a mean plasma concentration of SAHA capable of inhibiting a histone deacetylase in vivo in a subject over a period of si least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof) and a pharmaceuticaliy acceptable carrier or diluent. 10 Furthermore, the present invention provides a method of inhibiting fhe activity of a histone deacetylase in a subject, comprising producing a mean plasma concentration of at least 10 nM of SAHA in vivo in a subject over a period of at least two hours following administration by administering to said subject an effective amount of a pharmaceutical composition comprising SAHA or a pharmaceuticaliy acceptable salt or hydrate thereof 15 and a pharmaceuticaliy acceptable carrier or diluent in another preferred embodiment, the composition provides a mean plasma concentration of the HDAC inhibitor (e.g., SAHA) capable of itVhihitmg histone deacetylase over a period of at least 2 hours following administration, which is preferably at a concentration of at least about 10 nM. In another embodiment, the composition 20 provides a mean plasma concentration of the HDAC inhibitor of at least about 10 nM over a period of at least 8 hours following administration. In yet another preferred embodiment, the composition provides a mean plasma concentration of fhe HDAC inhibitor of at least about 10 nM over a period of at least 10 hours following administration. In yet another preferred embodiment, the composition provides a mean 25 plasma concentration of the HDAC inhibitor of at least about 10 nM over a period of at least 12 hours following administration. Non-limiting examples of mean plasma concentrations are about 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM, 1 jiM, 2 pM, 2.5 pM, 5 pM 10 fiM, 25, (xM, 50 pM, 100 nM and the like. It should be apparent to a person skilled in fhe art that these doses are in no way limiting the scope of this invention, and 30 that any mean plasma concentration which is capable of inhibiting a histone deacetylase is suitable. In one preferred embodiment, the composition provides a mean plasma concentration of fhe HDAC inhibitor (e.g., SAHA) capable of selectively inducing 19 WO 03/075839 568678 PCT/US03/0&451 tormina! fKffi»xfirifiarinp cell gTOWtfi SITBSt Eld/or apoptosis of neoplastic cells, or inducing differentiation of tumor cells in a tumor, wherein the concentration is maintained over a period of at least 2 hoars following administration, which is preferably at a concentration of at least about 2.5 pM. In another embodiment, the composition provides a mean. 5 plasma concentration of the HDAC inhibitor of at least about 25 fiM over a period of at least 8 hours following administration. In yet another preferred embodiment, fee composition provides a mean plasma concentration of the HDAC inhibitor of at least about Z.5 fiM over a period, of at least 10 hours following administration. la yet another preferred embodiment, the composition provides a mean plasma concentration of the 10 ' HDAC inhibitor of at least about 2.5 fiM over a period of at least 12 hours following administration. Non-limiting examples of mean plasma concentrations are about 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM. 1 pM, 2 jiM, 2.5 fiM, 5 fiM 10 pM, 25, pM, 50 jiM, 100 nM and the like. It should be apparent to a person skilled in the art feat these doses are in no way limiting the scope of this invention, and that any mean plasma 15 concentration which is capable of inducing terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells is suitable. In another preferred embodiment, the composition provides a mean plasma concentration, of the HDAC inhibitor (e.g^ SAHA) effective to induce differentiation of tumor cells in a subject having a tumor, wherein the amount is maintained for a period of 20 at least 2 hours following administration to the subject, in another preferred embodiment, the composition provides a mean plasma concentration of fhe HDAC inhibitor effective to induce differentiation of tumor cells in a subject having a tumor, wherein the amount is maintained for a period of at least 8 hours following administration to the subject In another preferred embodiment, the composition provides a mean plasma concentration of 25 the HDAC inhibitor effective to induce differentiation of tumor cells in a subject having a tumor, wherein the amount is maintained for a period of at least about 10 hours following administration to fhe subject Non-limiting examples of mean plasma concentrations are about 10 nM, 25 nM, 40 nM, 45 nM, 50 nM, 100 nM, 1 nM, 2 pM, 2.5 jjM, 5 pM 10 fiM, 25, pM, 50 fiM, 100 fiM and tie like. It should be apparent to a person skilled in the art 30 that these doses are in no way limiting the scope of this invention, and that any mean plasma concentration which is capable of inducing differentiation of tumor cells in a tumor is suitable. 20 WO 03/075839 568678 PCT/US03/0M51 The methods of fhe present invention are suitable for practice in vitro and in vivo. If the methods are practiced in vitro, contacting may be effected by incubating the cells with the compound. The concentration of fhe compound in contact with fhe cells should be from about 1 about nM to about 25 mM, far example, from about 10 nM to about 1 mM, 5 from about 40 nM to about 0.5 mM. Non-limiting examples of specific doses are 10 nM, 25 nM, 40nM, 45 nM, 50nM, 100 nM, 1 pM, 2 pM, 2.5 pM, 5 pM, 10 pM,25, pM, 50 pM, 100 fiM and ths like. The concentration depends upon the individual compound and the state of the neoplastic cells. Although the methods of fhe present invention can be practiced in vitro, it is 10 contemplated that the preferred embodiment for fhe methods of selectively manning terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells will comprise contacting the cells in vivo, Le., by administering the compounds to a subject harboring neoplastic cells or tumor cells in need of treatment The methods of the present invention may also comprise initially administering to 15 the subject an antitumor agent so as to render the neoplastic cells in the subject resistant to an antitumor agent and subsequently administering an effective amount of any of the compositions of the present invention, effective to selectively induce terminal differentiation, cell growth arrest and/or apoptosis of such cells. The antitumor agent may be one of numerous chemotherapy agents such as an 20 alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, colchicine, a vinca alkaloid, L-asparaginass, procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide. Suitable agents are those agents that promote depolarization of tubulin. Preferably the antitumor agent is colchicine or a vinca alkaloid; especially preferred are vinblastine and vincristine. In embodiments where the antitumor agent is 25 vincristine, the cells preferably are treated so that they are resistant to vincristine at a concentration of about 5 mg/ml. The treating of the cells to render them resistant to an antitumor agent may be effected by contacting the cells with the agent for a period of at least 3 to 5 days. The contacting of the resulting cells with any of the compounds above is performed as described previously. In addition to the above chemotherapy agents, the 30 compomids may also be administered together with radiation therapy. The present invention also provides a method of treating a patient having a tumor characterized by proliferation of neoplastic cells which comprises administering to the patient an effective amount of any of the compositions of the present invention above, 21 WO C3/075K39 568678 PCT/DS03/06451 effective to selectively induce terminal differentiatioE of such neoplastic cells and thereby inhibit their proliferation. The method of fhe present invention is intended for the treatment of human patients with .tumors. However, it is also likely that the method would be effective in the 5 treatment of tumors in other mammals. The teim tumor is intended to include any cancer caused by fhe proliferation of neoplastic cells, such as lung cancer, acute lymphoid myeloma, Hodgkms lymphoma, non-Hodgkins lymphoma, bladder melanoma, renal carcinoma, breast carcinoma, prostate carcinoma, ovarian carcinoma or colorectal carcinoma. - , 10 The administration of the pharmaceutical compositions can be carried out in unit dosages which may be administered orally once a day, twice a day, three times a day and the like. Currently preferred embodiments are once-daily administration, twice-daily administration and three-times daily administration. 15 Histone Deacetvlases aad Histone Deacetylase inhibitors Histone deacetylases (HDACs), as that term is used herein, are enzymes that catalyze the removal of acetyl groups from lysine residues in the amino tenninal tails of the nucleosomal core histones. As such, HDACs together with histone acetyl transferases (HATs) regulate fhe acetylation status of histones. Histone acetyiation affects gene 20 expression and inhibitors of HDACs, such as the hydroxamic acid-based hybrid polar compound suberoylanilide hydroxamic acid (SAHA) induce growth arrest, differentiation and/or apoptosis of transformed cells in vitro and inhibit tumor growth in vivo. HDACs can be divided into three classes based on structural homology. Class I HDACs (HDACs 1,2, 3 and 8) bear similarity to the yeast RPD3 protein, are located in fhe nucleus and are 25 found in complexes associated with transcriptional co-repressors. Class H HDACs (HDACs 4,5, 6,7 and 9) are similar to the yeast HDA1 protein, and have both nuclear and cytoplasmic subcellular localization. Both Class I and II HDACs are inhibited by hydroxamic acid-based HDAC inhibitors, such as SAHA Class H HDACs form a structurally distant class of NAD dependent enzymes that are related to fhe yeast SIR2 30 proteins and are not inhibited by hydroxamic acid-based HDAC inhibitors. Histone deacetylase inhibitors or HDAC inhibitors, as that term is used herein are compounds that are capable of inhibiting fhe deacetylation of histones in vivo, in vitro or both. As such, HDAC inhibitors inhibit the activity of at least one histone deacetylase. As a result of inhibiting the deacetylation of at least one histone, an increase in acetylated 1 22 WO 63/075839 568678 PCT/DS03/06451 histone occurs and accumulation of acctylated histone is a suitable biological marker for assessing the activity of HDAC inhibitors. Therefore, procedures that can assay for ths accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of compounds of interest B is understood that compounds that can inhibit histone 5 deacetylase activity can also bind to other substrates and as such can inhibit other biologically active molecules such as enzymes. It is also to be understood that the compounds of the present invention are capable of inhibiting any of fhe histone deacetylases set forth above, or any other histone deacetylases. For example, in patients receiving HDAC inhibitors, the accumulation of 10 acetylated histones in peripheral mononuclear cells as well as in tissue treated with HDAC inhibitors can be determined against a suitable control. HDAC inhibitory activity of a particular compound can be determined in vitro nginp for example, an enzymatic assays which shows inhibition of at least one histone deacetylase. Further, determination of fhe accumulation of acetylated histones in cells 15 treated with a particular composition can be determinative of the HDAC inhibitory activity of a compound. Assays for the accumulation of acetylated histones are well known in the literature. See, for example, Marks, P.A. et al., J. Natl. Cancer Inst, 92:1210-1215, 2000, Butler, LM. et al., Cancer Res. 60:5165-5170 (2000), Richon, V. M. et al., Proc. Natl. Acad. ScL, 20 USA, 95:3003-3007, 1998, and Yoshida, M. et al., J. Biol. Chan., 265:17174-17179, 1990. For example, an enzymatic assay to determine the activity of a histone deacetylase inhibitor compound can be conducted as follows. Briefly, the effect of an HDAC inhibitor compound on affinity purified human epitope-tagged (Flag) HDAC1 can be assayed by 25 mrpharing the enzyme preparation in the absence of substrate on ice for about 20 minutes ■with the indicated amount of inhibitor compound. Substrate fl^HJacetyl-labelled murine erythroleukemia cell-derived histone) can be added and the sample can be incubated for 20 minutes at 37°C in a total volume of 301jL. The reaction can then be stopped and released acetate can be extracted and the amount of radioactivity release determined by scintillation 30 counting. An alternative assay useful for determining the activity of a histone deacetylase inhibitor compound is the "HDAC Fluorescent Activity Assay; Drug Discovery Kit-AK-500" available from BIOMOL® Research Laboratories, Inc., Plymouth Meeting, PA. In vivo studies can be conducted as follows. Animals, for example, mice, can be injected intraperitoneally with an HDAC inhibitor compound. Selected tissues, for 568678 WO 03/075839 PCT/LTS03/064S3 example, twain. spleen, liver etc, can be isolated at prsdetsnninsd times, post artmini strati fin. Histonss can be isolated from tissues essentially as described, by Yoshida ef al., J. BioL ChenL 265:17174-17179,1990. Equal amounts of histones (about 1 fig) can bs electrophoressd on 15% SBS-polyaarylamide gels and can be transferred to Hybond-P 5 filters (available from. Amsrsham). Filters can be blocked with 3% milk aad can be probed ■with a rabbit purified polyclonal anti-acetylated histone H4 antibody (aAc~H4) and anti-acetyiated histone S3 antibody (aAc-H3) (Upstate Biotechnology, Inc.). Levels of acetylated histone can be visualized using a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5000) and the SuperSignal cfaernihtTnmesceait substrate (Pierce). As a 10 loading control for fee histone protein, parallel -gels can be ran and stained with Coomassie Blue (CB). hi addition, hydroxamic acid-based HDAC inhibitors have been shown to up regulate the expression of the P21™*1 gene. He p21wri protein is induced within 2 horns of culture with HDAC inhibitors in a variety of transformed cells using standard methods. 15 The induction of the p21wn gene is associated with accumulation of acetylated histones in ■the chromatin region of this gene. Induction of p21w» can therefore be recognized as involved in ths G1 cell cycle arrest caused by HDAC inhibitors in transformed cells. Typically, HDAC inhibitors fell into five general classes: 1) hydroxamic acid derivatives; 2) Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 20 and 5) electrophilic ketones. Thus, the present invention includes within its broad scope compositions comprising HDAC inhibitors which are 1) hydroxamic acid derivatives; 2) Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and/or any other class of compounds capable of inhibiting histone deacetylases, for use in 25 inhibiting histone deacetylase, inducing terminal differentiation in neoplastic cells, and /or inducing differentiation of tnmor cells in a tumor. Examples of such HDAC inhibitors include, but are not limited to: A. Hydra-ramie Acid Derivatives such as suberoylanilide hydroxamic acid (SAHA) (Richon et aL, Proc. Natl. Acad. Sci. USA 95,3003-3007 (1998)); m-carboxycinnamic 30 add bishydroxamide (CBHA) (Richon et al., supra); pyioxamide; trichostatin analogues such as trichostatin A (TSA) and trichostatin C (Koghe et al. 1998. Biochem. Pharmacol. 56: 1359-1364); salicylihydraxamic acid (SBHA) (Andrews et al., International J. Parasitology 30,761-768 (2000)); suberoyi bishydroxamic acid (SBHA) (U.S. Patent No. 5,608,108); azelaic bishydroxamic acid (ABHA) (Andrews et aln supra); azelaic-1- 568678 WO 03/075839 PCT/CS03/GS451 hydroxamate-9-artiIide (AAHA) (Qiu et ed., Mol. Biol. Ceil 11, 2069-20E3 (2000)); 6-(3~ chloropheiiylureido) caipoic 'cydroxamic acid (3C1-UCHA); oxamfiatm [(2E)-5-[3-[(phenylsufonyl) aminol ^eayl]-psQt-2^^ynohyiiroxaimc acid] (Kim et al Oncogene, 18: 2461 2470 (1999)); A-161906, Soriptaid (Su et al. 2000 Cancer Research, 60: 3137-5 3142); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews et al., supra); MW2996 (Andrews et al., supra); or any of fhe hydroxamic acids disclosed in U.S. Patent Numbers 5,369,108,5,932,616,5,700,811,6,087,367 and 5,511,990. B. Cyclic Tetrapeptjdes such, as traporm A (TPX}-cyclic tetrapsptide (cyclo-(L-10 pbenylakDyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-S-oxo-9;10-epoxy decanoyl)) (Kijima et al., J BioL Chem. 268,22429-22435 (1993)); ER901228 (FK 228, depsipeptide) (Nakajima et al., Ex. Cell Res. 241,126-133 (1998)); FR225497 cyclic tetrapeptide (H. Mori et al., PCT Application WO 00/08048 (17 February 2000)); apicidin cyclic tetrapeptide [cyclo(N-0-mefiiyi-L-tryptophanyl-L -isoleuciayl-E)-pipecolinyl-L-2-Ernino-15 8-oxodecanoyl)] (Daridn-Rattray et al., Proc. Natl. Acad. ScL USA 93,1314313147 (1996)); apicidin la, apicidin lb, apicidin Ic, apicidin Ha, and apicidin Hi (P. Dulski et al., PCT Application WO 97/11366); CHAP, HC-toxin cyclic tetrapeptide (Bosch et al., Plant Cell 7, 1941-1950 (1995)); WF27082 cyclic tetrapeptide (PCT Application WO 98/48825); and chlamydocin (Bosch af al., snpra). 20 C. Short chain fatty acid fSCFA") derivatives such as: sodium butyrate (Couseos et al., J. Biol. Chem;'254,1716-1723 (1979)); isovalerate (McBain et al., Biochem. Phann. 53: 1357-1368 (1997)); valerate (McBain et al., supra) ; 4-phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)); 25 phenylbutyrate (PB) (Wang et a/., Cancer Research, 59, 2766-2799 (1999)); propionate (McBain et al., supra); butyramide (Lea and Tulsyan, supra); isobutyramide (Lea and Tulsyan, supra); phenylacetate (Lea and Tulsyan, supra); 3-bromopropionate (Lea and Tulsyan, supra); tributyrin (Guan et al., Cancer Research, 60,749-755 (2000)); valproic add and valproate. 30 D. Benzamide derivatives such as CI-994; MS-27-275 [N- (2-aminophenyi)-4- [N-(pyridin-3-yl msfhoxycaibonyl) ammomethyl] bsnzamide] (Saito et al., Proc. Natl. Acad. Sd. USA 96, 4592-4597 (1999)); aad 3'-aaimo derivative of MS-27-275 (Saito et ah, supra). 25 568678 WO 03/075839 PCT7US03/06451 E. Electrophflic ketone derivatives sudi as Mfiuoramethyl ketones (Prey et al, Bioarganic & Med. Cham. Lett (2002), 12, 3443-3447; U.S. 6.511,990) and a-keto amides such, as N-mefbyl- a-ketoatnides 5 F. Other HDAC Inhibitors such as depudecin (Kwoq et al. 1998. PNAS 95: 3356-3361. Preferred hydroxamic acid "based HDAC inhibitors are suberoylanilide hydroxamic 10 acid (SAHA), m-carboxycinnainic acid bishydroxamaie (CBHA) and pyroxamide. SAHA has been shown to bind directly in the catalytic pocket of the histone deacetylase enzyme. SAHA induces cell cycle arrest, differentiation and/or apoptosis of transformed cells in culture and inhibits tumor growth, in rodents. SAHA is effective at inducing these effects in both solid tumors and hematological cancers. It has been shown that SAHA is effective 15 at inhibiting tumor growth in animals with no toxicity to the animal. The SAHA-indnced inhibition of tumor growth is associated with an accumulation of acetylated histones in the tumor. SAHA is effective at inhibiting the development and continued growth of carcinogen-induced (N-methylnitrosourea.) mammary tumors in rats. SAHA was administered to the rats in their diet over the 130 days of the study. Thus, SAHA is a 20 nontoxic, orally active antitumor' agent whose mechanism of action involves the inhibition of histone deacetylase activity. Preferred HDAC inhibitors are those disclosed in U.S. Patent Numbers 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511, 990, issued to some of the present inventors disclose compounds, the entire contents of which are incorporated herein by reference, 25 non-Emiting examples of which are set forth below: Thus, in one embodiment, the present invention provides a pharmaceutical composition comprising a compound presented by the structure of formula 1, or a pharmaceuticaliy acceptable salt or hydrate thereof) and a pharmaceuticaliy acceptable carrier or excipient *\ / V—(CH2)n— 30 0 Ra CD wherein R) and R2 can be fhe same or different; when Ri and R2 are fhe same, each is a 26 568678 WO 03/C75839 PCT/DS03/0&451 substituted or unsubstitiited arylanmo, cyclaalkylamina, pyridineamino, pipgridiao, 9-pizriiie-6-aimne or feia2oleainiao group; whan Rj and Ri are different Rj=Pc--N-K.4, wherein each of Sj and Ra are independently the same as or different from each ofosr and ar® a hydrogen atom, a hydroxyl group, a substituted or unsubstituted. branched or unbranched 5 alkyl, alkenyl, cycloalkyl, aryl alkyloxy, aryioxy, aryialkylaxy or pyridine group, or Rj and R< are bonded together to form a piperidins group, R* is a hydroxylainino, hydroxyl, amino alkylamino, dialkjiamiao or alkyloxy group and n is an integer from about 4 to about S. In a particular embodiment of Fcamula 1, Ri and R2 are fhe same and. are a 10 substituted or unsubstituted thiazoleaxoiao group; and n is an integer from about 4 to about 8. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 2, or a pharmaceuticaliy acceptable salt or hydrate thereof) and a pharmaceuticaliy acceptable 15 carrier or excipient. /* \ / C (CHj)n 0 Rj (2) wherein each of Rj and R* are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted, branched or 20 uobranched aScyi, aJkenyl, cycloalkyl, arylalkyloxy, ■ aryioxy, arylalkyloxy or pyridine group, or Rs and R4 are bonded together to form a piperidine group, R2 is a hydroxyiamino, hydroxy!, amino, alkylammo, dialkylamino or alkyloxy group and n is an integer from about 4 to about 8. In a particular embodiment of formula 2, each of R3 and R4 are independently the 25 same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted, branched or tmbranched alkyl, alkenyi, cycloalkyl, aryl, alkyloxy, aryioxy, arylalkyloxy, or pyridine group, or R3 and R4 bond together to form a piperidine group; Rj is a hydroxyiamino, hydroxy!, amino, alkykmino, or alkyloxy group; n is an integer from 5 to 7; and R3-N-R4 andR.2 are different 30 In another particular embodiment of Formula 2, n is 6. In yet another embodiment of Formula D, R« is a hydrogen atom, R3 is a substituted or unsubstituted phanyi and n is 6. 27 568678 WO 03/075839 PCT/US03/06451 In yet another embodiment of Formula IE, R* is a hydrogen atom, R3 is a substituted phenyl and 11 is 6, wherein fhe phenyl suhstrtuent is selected from ths group consisting of a mefhyl, eyano, nitro, ttifluoromeihjd, amino, aminocarbonyl, mesfhylcyaiio, chloro, fhioro, bromo, iodo. 2,3-difluoro, 2,4-diflaoro, 2^-difkiaro, 3,4-dLSuoro, 3,5-di£hioro, 2,6-5 difluaro, 1,2,3-trifluaro, 2,3,6-tdfiuoro, 2,4,6-trifiuoro, 3,4,5-trifluoro, 2,3,5,6-tstrafluoro, 2,3,4,5,6-pentafl.uoro, azido, hexyl, t-butyL phenyl, carbaxyl, hydroxyl, methoxy, phenyloxy, benzyloxy, phenylarainooxy, phsnylaminocarbonyl, methoxycarbonyl, mdhyiammocarbonyl, dimethyiamino, dimethylamiiio carbonyl, or hydrosyiaminocarbonyl group. 10 In another embodiment of formula 2, a is 6, R$ is a hydrogen atom and R3 is a cyclohexyi group, ia another embodiment of formula 2, n is 6, R4 is a hydrogen atom and R3 is a methoxy group. In another embodiment of formula 2, n is 6 aad R3 and R4 "bond together to form a piperidine group. In another embodiment of formula 2, n is 6, R* is a hydrogen atom and H3 is a benzyioxy group. In another embodiment of formula 2, R4 is a 15 hydrogen atom and R3 is a ^-pyridine group, hi another embodiment of formula 2, R4 is a hydrogen atom and R3 is a ^-pyridine group. In another embodiment of formula 2, R4 is a hydrogen atom and R3 is an a-pyridme group. In another embodiment of formula 2, n is 6, and R3 aad R* are both methyl groups. Ia another embodiment of formula H, a is 6, R4 is a methyl group and R3 is a phenyl group. 20 In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 3, or a •A >'.• •' pharmaceuticaliy acceptable salt or hydrate thereof; and a pharmaceuticaliy acceptable carrier or excipient. 25 wherein a is an integer from 5 to about 8, 30 In a preferred embodiment of formula 3, a is 6. In accordance with this embodiment, the present invention provides a pharmaceutical composition comprising SAHA (4), or a pharmaceuticaliy acceptable salt or hydrate thereof, and a 28 WO C3/C75E3S 568678 PCT/US03/06451 10 15 phairoaceaifeally acceptable carrier or excipient SAHA can be represented by fits following structural formula. (4) In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 5, or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or excipient. CK N Jj 0 ,c (ch2)6 / o nhoh (5) fa. another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 6 (pyroxamide), or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or excipient h \ / c (ch2}6 & 20 0 NHOH (6) la another embodiment, the present invention provides a pharmaceutical 25 composition comprising a compound represented by the structure of formula 7, or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or excipient 29 568678 WO C3/075839 PCT/US03IOM51 -(CH2)e i ,0 \f-IOH (7) In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 8, or a phannaceatioally acceptable salt or hydrate thereof, and a phaxmaceutically acceptable 10 carrier or exripient. O (CH2)e {/ nhoh 15 (8) In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 9, or a 20 pharmaceuticaliy acceptable salt or hydrate thereof) aad a pharmaceuticaliy acceptable canier or excipient H / \ I If v> CH2 N, 25 \—(CH2)6—// o nhoh (9) Si another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by fee structure of formula 10, or a pharmaceuticaliy acceptable salt or hydrate thereof) and a pharmaceuticaliy acceptable carrier or excipient 30 568678 WO 03/075839 PCT/C503/0M51 0 r2 (10) 5 wherein R3 is hydrogen and K4 cycloalkyl, aryl, aryioxy, arylalkyloxy, or pyridine group, or R.3 and R4 bond together to form a piperidine group; R2 is a hydroxyiamino group; and n is an integer from 5 to about 8. In. another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 11, or a 10 phatmaceutically acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or excipient wherein R3 and R4 are independently a substituted or unsubstituted, branched or unbranched alkyi, alkenyl, cycloalkyl, aryl, alkyloxy, aryioxy, arylalkyloxy, or pyridine group, cycloaBcyl, aryl, aryioxy, arylalkyloxy, or pyridine 'group, or R3 and R4 bond' together to form a piperidine group; R2 is a hydroxyiamino group; and n is an integer 20 from 5 to about 8. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 12, or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or excipient, 01) 15 25 'Y X . R (12) 31 WO 03/075839 568678 PCT/US03AW51 wherein each of X and Y are independently fhe same as or different from each, other and are a hydroxyl, amWi or hydroxyiamino group, a substituted or unsubstitated alkyloxy, alkylamino, diaBcylamino, arylamino, alkyiarylamirto, alkylcxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group; R is a hydrogen atom, a hydroxyl, 5 group, a substituted or unsubstituted alkyl, arylalkyloxy, or aryioxy group; and each of m and n are independently the same as or different from each other and are each an integer from about 0 to about 8. In a particular embodiment, the HDAC inhibitor is a compound of Fonnula XI wherein X, Y and R are each hydroxyl and both m and n are 5. 10 In another embodiment, the present indention provides a pharmaceutical composition comprising a compound represented by the structure of formula 13, or a pharmaceuticaliy acceptable salt or hydrate thereof) and a pharmaceuticaliy acceptable carrier or excipient. n O o \ ii ii y (H2C)m C N ,(OH2)n N C (Cl-yc- c' I I 15 (13) wherein each of X aad Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxyiamino group, a substituted or unsubstituted alkyloxy, aflcyiamino, dialkylamino, arylamino, alkylarylamiio, alkyloxyamino, aryloxyamino, 20 alkyioxyalkylammo or aryloxyalkylamino group; each of Rj and Rz are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl, aryl, alkyloxy, or aryioxy group; and each of m, n and o are independently the same as or different from each other and are each an integer from about 0 to about 8. 25 In one particular embodiment of fonnula 13, each of X and Y is a hydroxyl group and each of Ri and Ra is a methyl group. In another particular embodiment of formula 13, each of X and Y is a hydroxyl group, each of Ri and R2 is a methyl group, each of n and 0 is 6, and m is 2. In another embodiment, lite present invention provides a pharmaceutical 30 composition comprising a compound represented by the structure of formula 14, or a pharmaceuticaliy acceptable salt or hydrate thereof) and a phannaceutically acceptable 32 WO 03/075R39 568678 PCT7US03/06451 wherein each of X and Y are independently the same as or different from each other and 5 are a hydroxyl, amino or hydroxyiamino group, a substituted or unsubstituted alkyloxy, alVylamfnn. dialkylaxnino, arylamino, alkylarylamino, alkyloxyamino, aryloxyanimo, aByloxyalkylamino or aryloxyalkylamino group; each of Ri and R2 are independently the same as or different from each other and axe a hydrogen atom, a hydroxy! group, a . substituted or unsubstituted alkyl, aryl, alkyloxy, or aryioxy group; and each of m and n 10 are independently flue same as or different from each other and are each an integer from about 0 to about 8. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 15, or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable 15 carrier or exripient (15) 20 wherein each of X and Y are independently fhe same as or different from each other and are a hydroxyl, amino or hydroxyiamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, aliylarylammo, alkyloxyamino, aryloxyamino, alkyloxyalkylamino or aryloxyalkylamino group; and each of m and n are independently the same as or different from each other and are each an integer from about 0 to about 8. 25 In one particular embodiment of formula 1, each of X and Y is a hydroxyl group and each of m and n is 5. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 16, or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable 30 carrier or excipient 33 568678 WO 03/075839 PCT70S03/06451 -(h2C)l > wherein each of X and Y are independently fhe same as or different from each, other and 5 are a hydroxyl, amino or hydroxyiamino group, a substituted or unsubstituted alkyloxy, aDcyiamino, dialiyiamino. aiylamrio, alkylaiylamino, alkyloxyamino, aiyloxyaroino, alkyloxyaJkyiamino or aryloxyalkylamino group; Ri and R2 are independently the gam* as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl, arylalkyloxy or aryioxy group; and each of m and n are independently 10 the same as or different from each ofhex and are each ah integer from about 0 to 'about 8. En another embodiment, fhe present invention provides a pharmaceutical composition comprising a compound represented by fhe structure of fonnula 17, or a phaimaceutically acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable carrier or excipienL CH3 CH3 O 15 X c CH (CHj)n CH— (17) wherein each of X an Y are independently lie same as or different from each other and are a hydroxy!, amino or hydroxyiamino group, a substituted or unsubstituted aikyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, or aryloxyaflcylamino group; and n 20 is an integer from about 0 to about 8. hi one particular embodiment of formula 17, each of X and Y is a hydroxyiamino group; Ri is a methyl group, R2 is a hydrogen atom; and each of m and n is 2. in another particular embodiment of formula 17, each of X and Y is a hydroxyiamino group; Ri is a carbonylhydroxylamino group, R2 is a hydrogen atom; and each of m and a is 5. in 25 another particular embodiment of formula 17, each of X and Y is a hydroxyiamino group; each of Ri and R3 is a fiuoro group; and each of m and n is 2. In another embodiment, fhe present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 18, or a pharmaceuticaliy acceptable salt or hydrate thereof, aid. a pharmaceuticaliy acceptable 30 carrier or excipienL 34 WO G3/B75839 568678 pcrrosasfOMsi ir H> r i x c (CH2)m C (CH^it C—Y R2 (18) wherein each of X and Y are independently &e same as or different from each, other and 5 are a hydroxy!, aminn 0r hydroxyiamino group, a substituted or unsubstituted alkyloxy, alkylsmino, dialkylamino, aiyiamino, alkylarylamino, alkyloxyamino, aiyloxyamiao, alkyloxyalkyamino or aiyloxyalkylamiiio group; each of Rj and E2 are independently the same as or different from, each other and are a hydrogen, atom, a hydroxy! group, a substituted or unsubstituted alkyl, aryl, alkyloxy, aryioxy, carbonylhydroxylamino or 10 fiuoro group; and each of m and n are independently the same as or different from each other and are each an. integer from about 0 to about 8. In. another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 19, or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable 15 carrier or excipienL wherein each of Rj and R2 are independently the same as or different firom each other and 20 are a hydroxyl, alkyloxy, amino, hydroxyiamino, alkylamino, dialkylamino, aiyiamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group. In a particular embodiment, the HDAC inhibitor is a compound of structural Fonnula X wherein Ri and Ri are both hydroxyiamino. 1 In one particular embodiment of fonnula 19, Ri is a phenylamino group and Rj is a 25 hydroxyiamino group. Ia another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 20, or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable 35 WO 03/075839 568678 PCTATS03/06451 carrier or excipient. (20) 5 wherein each, of Rt and Ri are independently fee same as or different from each other and are a hydroxy!, alkyloxy, amino, hydroxylammo, alkylamino, dialkylamino, aiyiamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group. In a particular embodiment, fhe HDAC inhibitor is a compound of structural Fonnula XI wherein Ri and Ri are both hydroxyiamino. 10 In one particular embodiment of formula XVHI, Ri is a hydroxyiamino group. In another particular embodiment of formula 21, Rz is a hydroxyiamino group. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of fonnula 22, or a pharmaceuticaliy acceptable salt or hydrate thereof; and a pharmaceuticaliy acceptable 15 carrier or excipient. wherein each of Ri and R2 are independently fhe same as or different from each other and are a hydroxy!, alkyloxy, amino, hydroxyiamino, alkylamino, dialkylamino, arylamino, 20 alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group. In a particular embodiment, the HDAC inhibitor is a compound of structural Formula XII wherein Ri aad R2 are both hydroxyiamino. In one particular embodiment of fonnula 23, Ri is a phenyl amino group and Rj is a hydroxyiamino group. 25 In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of fonnula 24, or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or excipient. (22) 36 WO 03/075839 568678 PCT/US03/064SI R 0 R (24) wherein R is a phenylamino group substituted wilt a cyano3 methylcyano, nitro, carboxyl, 5 amittocarbcmyl, mefhyiarainocarbonyi, dimetbylaminocafbonyl, trifhioromefhyl, hyaroxylaminocarbonyl, N-bydroxylaminocarbonyL methoxycarbonyl, chloro, fluoro, methyl, msfhaxy, 2,3-diflaaro, 2,4-difluoro, 2,5-difluoro, 2,6-difiiloro, 3,5-diQaoro, 2,3,6-trifluoro, 2.4,6-trifluoro, 1,2,3-trifLuoro, 3,4,5-trifhioro, 2,3,4,5-tetrafhioro, ar 2,3,4.5,6-pentafluoro group; and n is an integer fiom 4 to 8. 10 In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 25 (CBHA), or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceutical!y acceptable carrier or excipient composition comprising a compound represented by the structure of formula 26, or a 20 pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or excipient 15 o (25) hi another embodiment, the present invention provides a pharmaceutical o R1 c_, H :CH (26) 25 In another embodiment, the present invention provides a pharmaceutical 37 568678 WO 03/075839 PCT/DS03/06451 composition comprising a compound represented by fhe structure of formula 27, or a pharmaceuticaliy acceptable salt or hydrate thereat and a pharmaceuticaliy acceptable carrier or excipient, 5 R c NH (CHjJn C NHOH (27) wherein R is a substituted or unsubstituted phenyl, piperidine, thiazole, 2-pyiidiae, 3-pyridine or 4-pyridine and n is an integer from about 4 to about 8. In one particular embodiment of formula 27, R is a substituted phenyl group. In 10 another particular embodiment of formula 27, R is a substituted phenyl group, where the substituent is selected from the group consisting of methyl, cyano, nitro, fhio, trifluoromefhyl, amino, aminocarbonyl, mefiiyicyaao, chloro, fluoro, bromo, iodo, 2,3-diftiioro, 2,4-difluoro, 2,5-difluoro, 3,4-difluoro, 3,5-difiuoro, 2,5-difluoro, 1,23-trifluoro, 2,3,6-trifluaro, 2,4,6-trifhaoro, 3,4,5-trifluoro, 23,5,5-tefrafluoro, 2,3,4,5,6-pemafluoro, 15 azido, hexyl, t-butyi, phenyl, carboxyl, hydroxyl, methyloxy, phenyloxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methyioxycarbonyl, methyiam in ocarbonyl, dimethylamino, dimethylaminocarbonyl, or hydroxylaminocarbonyl group. In another particular embodiment of formula 27, R is a substituted or unsubstituted 2-pyridine, 3-pyridine or 4-pyridine and n is an integer from about 4 to about 8. 20 In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 28, or a pharmaceuticaliy acceptable salt or hydrate thereof and a pharmaceuticaliy acceptable carrier or excipient o p R HN C NH (CHjdn c—NHOH 25 (28) wherein R is a substituted or unsubstituted phenyl, pyridine, piperidine or thiazole group and n is an integer from about 4 to about S or a pharmaceuticaliy acceptable salt thereof hi a particular embodiment of formula 28, R is a substituted phenyl group. Ia another particular embodiment of formula 28, R is a substituted phenyl group, where the 30 substituent is selected from fhe group consisting of methyl, cyano, nitro, fhio, trifluoromethyi, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2,3-difhioro, 2,4-difhioro, 2,5-difhioro, 3,4-difiuaro, 3,5-difluoro, 2,6-difhioro, 1,2,3-trifluaro, 38 WO 03/075839 568678 PCT/US03/0645I 2,3,6-trifluoro, 2,4,64riftuaro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4,5.6-penta£tuoro, szido, hexyl, t-bntyi, phenyl, earboxyl, hydroxyl, znafhyioxy, phsnyloxy, bsnzyloxy, phenylaminooxy, phsnyiaminocaibonyl, methyloxycafbonyl, mefhylaminocarbanyl, dimefhylamino, dimefoylammocarbonyl, or hydroxyiaminocafbonyl group. 5 In another particular embodiment of formula 28, R is phenyl and n is 5. In another embodimsni, n is 5 and R is 3-chlorophenyl. In another embodiment fhe present invention provides a pharmaceutical composition comprising a compound represented by fhe structure of formula 29, or a pharmaceuticaliy acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable 10 carrier or excipient wherein each of Ri and R2 is directly attached or through a linker and is substituted or unsubstituted, aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, 15 cycloalkylamino, pyridmeanuno, piperidine, 9-purine-6-amiiio, thiazoleamitio, hydroxyl, branched or unbranched alkyl, alkeayl, alkyloxy, aryioxy, arylalkyloxy, pyridyl, or quinolinyl or isoquinolinyl; n is an integer from about 3 to about 10 and R3 is a hydroxamic acid, hydroxyiamino, hydroxyl, amino, alkylamtno or alkyloxy group. The linker can be an amide moiety, e.g., 0-, -S-, -NH-, NR5,-GELs-, -(CH^ni-, -(CEEKIH)-, 20 phenylene, cycloalkylene, or any combination thereof, wherein Rs is a substitute or unsubstituted C1-C5 alkyl. In certain embodiments of formula 29, Ri is -NH-Rt wherein R* is substituted or unsubstituted, aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridineamino, piperidine, 9-purine-6-amino, thiazoleamino, hydroxyl, 25 branched or uribranched alkyl, alkenyl, alkyloxy, aryioxy, arylalkyloxy, pyridyl, quinolinyl or isoquinoliayl In another embodiment, the present invention provides a pharmaceutical composition comprising a compound represented by the structure of formula 30, or a phannaceutically acceptable salt or hydrate thereof, and a pharmaceuticaliy acceptable 30 carrier or excipient 39 WO 03/075839 568678 PCT/OS03/06451 0 *2 (30) wherein each of Ri and Rs is, substituted or unsubstituted, aryi (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridineamino, piperidioo, 9-purine-5 ft-amfnn. thiazoleamino, hydroxyl, branched or unbranched alkyl, alkenyi, alkyloxy, aryioxy, arylalkyloxy, pyridyl, quinolinyl or isoquioolinyl; Rj is hydroxamic acid, hydioxylamino, hydroxy], amino, aikyfamino or alkyloxy group; R* is hydrogen, halogen, phenyl or a cycloalkyl moiety, and A can be the same or different and represents an amide ' moiety, 0-, -S-, -NH-, NRs, -CHa-, -{CHiV, -(CH=CH)-, pheayiene, cycloalkyieais, or 10 any combination thereof wherein Rs is a substitute or unsubstituted Ci-Cs alkyl; and n and m are each an integer from 3 to 10. In 'farther particular embodiment compounds having a more specific structure within the scope of compounds 29 or 30 are: A compound represented by the structure of formula 31: O 15 "T (31) wherein A is an amide moiety, Ri and Rz'are each selected fiom substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino, fhiazoleamino, aryioxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; 20 and n is an integer from 3 to 10. For example, the compound of formula 30 can have the structure 31 or 32: o 0=^ (chjw^nhoh Y > m^s> "Rj lis (31) (32) 25 wherein Ri, Rs and n have the meanings of Formula 30. 40 568678 WO 03/075839 A compound represented by the structure of fonnula 33: (CHz>n .NHOH PCT70S03/06451 wlisrein R.7is selected from substitated or nnsubstituted aryl (e.g., phenyl), arylalkyl (&.g., 5 bercyl), naphthyl, pyridineamino, 9-purine-5-arnino, fhiaroleammo, aryioxy, arylalkyloxy, pyridyl, quinolinyl or isoqninolinyl; n is an integer fiom 3 to 10 and Y is selected from.: A compound represented by the structure of formula 34: 10 R7V (Ct-yn _,NHOH -NH Y (34) wherein n is an integer from 3 to 10, Y is selected from 41 568678 WO 03/C75839 PCT/US03/0W51 o6c6c6c6 cowccyay c6c6oioi caco'co'-ccy and Rj' is selected from 5 A compound represented by the structure of fonnula 35: Rt'V. JL .(Chyn .NHOH T J .NH r (35) 10 aryl (e.g., phenyl), axyialkyl (e.g., benzyl), naphthyl, pyridineamino, 9-pHrine-6-amino, thiazoleamino, aiyloxy, arylalkyloxy, pyridyl, quinolinyl or isoqianolinyl; n is an integer from 3 to 10 aodRj' is selected from 42 WO O3/075K39 568678 PCT/US03/06451 A compound represented by the structure of fonnula 36: 10 15 wherein A is an amide moiety, Rj and R2 are each selected from substituted or ttasubstitated aryi {e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino, thiazoleamino, aiyloxy, arylalkyloxy, pyridyl, quinolinyl or isoqumolinyl; Rt is hydrogen, a halogen, a phenyl or a cycloalkyl moiety and n is an integer from 3 to 10. For example, the compound of formula 36 can have the structure 37 or 3 8: NHOH NHOH 37 wherein Ri, R2, R+ and n have the meanings of Fonnula 36. A compound represented by the structure of formula 39: 38 NHOH wherein L is a linker selected from the group consisting of an amide moiety, 0-, -S-, -NH- 43 WO €3/075839 568678 KT/US03/D6451 , m5, -CH:-, -(CH2)bi-} -(CH=GH}-, phenylene, cycloalkylene, or any combination thereof wherein. Rs is a substitute or unsubstituted Ci-Cs alkyl; and wherein each ofS.7 aad Rs are independently a substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-aminos thiazoleamino, aryioxy, arylalkyloxy, 5 pyridyl, quinolinyl or isoquinolinyl,, n is an integer from 3 to 10 and m is an integer from 0-10. Oflier HDAC inhibitors suitable for use in fhe invention include those shown in the (41) wherein n is an integer from 3 to 10 or an enantiomer. in one particular embodiment of formula 41, n=5. 44 WO 03/075839 568678 PCT/US03/06451 wherein n is an integer fiom 3 to 10 or an enantionier. Ia one particular embodiment of formula 42, ii=5. 5 •wherein n is an integer fiom 3 to 10 or aa enantioEQer. In one particular embodiment of 10 formula 43, n=5. 45 568678 WO C3/075S39 PCT/CSB3/06451 wherein it is an integer from 3 to 10 or an snaoftomer. In one particaiar embodiment of formula 44, n=5. <CH2)n^ .NHOH -5 (45) wherein n is an iategear from 3 to 10 or an enantiomer. In one particular embodiment of formula 45, n=5. 10 ,MHOH (46) wherein n is an integer from 3 to 10 or an enantiomer. 3n one particaiar embodiment of formula 46, n=5. 15 46 WO €3/075839 568678 PCT/US03/06451 wherein n is an integer from 3 to 10 or ait enantiomer. la one particular embodiment of 5 formula 47, n-5. wherein n is an integer from 3 to 10 or an enantiomer. In one particular embodiment of 10 formula 48, ie=5. WO 03/075829 568678 PCT/US03/06451 (49) wherein n is an integsr from 3 to 10 or an anantiomar. In one particular embodiment of formula 49, n=5. NHOH (50) wherein n is an integer from 3 to 10 or an enantiamer. In one particular embodiment of formula 50, n=5. rr 10 (51) wherein n is an integer from 3 to 10 or an enantiomer. In one particular embodiment of formula 51, n=5. 15 (52) 48 568678 WO C3/C75S39 PCT/US&3/06451 wherein n is an integer from 3 to 10 or an enantiomer. In one particular embodiment of fonnula 52, n=5. Other eramples of such compounds and other HDAC inhibitors can be found in U.S. Patent No. 5,369,108, issued on November 29, 1994, U.S. Patent No. 5,700,811, 5 issued on December 23, 1997, U.S. Patent No. 5,773,474, issued on June 30, 1998, U.S. Patent No. 5,932,616, issued on August 3, 1999 and U.S. Patent No. 6,511,990, issued January 28, 2003, all to Breslow et al.; U.S. Patent No. 5,055,608, issued on October 8, 1991, U.S. Patent No. 5,175,191, issued on December 29, 1992 and U.S. Patent No. 5,608,108, issued on March 4, 1997, all to Maries et al.\ as well as Yoshida, M., et al, 10 Bioassays 17, 423-430 (1995); Saito, A, et al, PNAS USA 96, 4592-4597, (1999); Fin-amai R, et al, PNAS USA 98 (1), 87-92 (2001); Komatsu, Y., et al., Cancer Res. 61(11), 4459-4466 (2001); So, GJEL, elaL, Cancer Res. 60, 3137-3142 (2000); Lee, B.I. et al., Cancer Res. 61(3), 931-934; Suzuki, T., et al, J. Med. Cham. 42(15), 3001-3003 (1999); published PCT Application WO 01/18171 published on March 15,2001 to Sloart-15 Tattering; Institute for Cancer Research and The Trustees of Columbia University; published PCT Application W002/246144 to Hoffrn ana-La Rochs; published PCT Application W002/22577 to Novartis; published PCT Application W002/30879 to Prolifix; published PCT Applications WO 01/38322 (published May 31, 2001), WO 01/70675 (published on September 27,2001) and WO 00/71703 (published on November 20 30, 2000) all to Methylgene, Inc.; published PCT Application WO 00/21979 published on October 8, 1999 to Fnjisawa Pharmaceutical Co., Ltd.; published PCT Application WO 98/40080 published on March 11, 1998 to Beacon Laboratories, L.L.C.; and Curtin M. (Current patent status of histone deacetylase inhibitors Expert Opin. Ther. Patents (2002) 12(9): 1375-1384 and references cited therein). 25 SAHA or any of the other HDACs can be synthesized according to the methods outlined in the Experimental Details Section, or according to the method set forth in U.S. Patent Nos. 5,369,108, 5,700,811, 5,932,616 and 6,511,990, the contents of which are incorporated by reference in their entirety, or according to any other method known to a person skilled in the art 30 This invention, in addition to the above listed compounds, is intended to encompass the use of homologs and analogs of such compounds. In this context, homologs are molecules having substantial structural similarities to the above-described compounds and analogs are molecules having substantial biological similarities regardless of structural similarities. 49 WO 03/07583? 568678 PCI7US03/0645I The invention also encompasses pharmaceutical compositions comprising pharmaceuticaliy acceptable salts of the HDAC inhibitors with organic and inorganic acids, for example, acid addition salts which may, for example, be hydrochloric acid, sulphuric acid, msthanesulphonic acid, fumade acid, maieic acid, succinic: add, acetic 5 acid, benzoic: acid, oxalic acid, citric acid, tartaric acid, carbonic acid, phosphoric add and Ihe like. Pharmaceutical ty acceptable salts can also be prepared from by treatment with inorganic bases, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyteniine, trimethylamine, 2-ethylammo efaanol, histidine, procaine, and the lite. 10 The invention also encompasses pharmaceutical compositions comprising hydrates of the HDAC inhibitors. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrats and the like. This invention also encompasses pharmaceutical compositions comprising any solid or liquid physical form of SAHA or any of the other HDAC inhibitors. For example, 15 The HDAC inhibitors can be in a crystalline form, ia amorphous form, and have any particle size. Ihe HDAC inhibitor particles may be micronized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form. 20 Pharmaceutical compositions The compounds of fhe invention, and derivatives, fragments, analogs, homologs pharmaceuticaliy acceptable salts or hydrate thereof can be incoiporated into pharmaceutical compositions suitable for oral administration, together with a pharmaceuticaliy acceptable carrier or excipienL Such compositions typically comprise a 25 therapeutically effective amount of any of the compounds above, and a pharmaceuticaliy acceptable carrier. Preferably, the effective amount is an amount effective to selectively induce tenninal differentiation of suitable neoplastic cells and less than an amount which causes toxicity in a patient Any inert excipient that is commonly used as a carrier or diluent may be used in the 30 formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosie material, an acrylate, or mixtures thereof. A preferred diluent is microcrystalline cellulose. The compositions may further comprise a disintegrating agent (e.g, croscarmellose sodium) and a lubricant (e.g., magnesium stearate), and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, 50 WO 83/075839 568678 PCT/OSD3/O6451 a surfactant, a soiubilizing agent, a plasticizer, ail emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or arty combination thereof. Furthermore, fhe compositions of the present invention may be in fhe form of controlled release or immediate release formulations. 5 One embodiment is a pharmaceutical composition for oral administration comprising a HDAC inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof, microcrystalline cellulose, croscarmellose sodium and magnesium stearate. Another embodiment has SAHA as fhe HDAC inhibitor. Another embodiment comprises 50-70% by weight of a HDAC inhibitor or a pharmaceuticaliy acceptable salt or hydrate thereof 10 20-40% by weight microcrystalline cellulose, 5-15% by weight croscarmellose sodium and 0.1-5% by weight magnesium stearate. Another embodiment comprises about 50-200 mg of a HDAC inhibitor. In one embodiment, fhe pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid 15 preparation. 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. In one embodiment of the present invention, the composition is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise in addition to the HDAC inhibitor active 20 compound and the inert carrier or diluent, a hard gelatin capsule. As used herein, "pharmaceuticaliy acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration, such as sterile pyrogen-free water. Suitable carriers are described in the 25 most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Hie use of such media and agents for pharmaceuticaliy active 30 substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into fhe compositions. Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., com starch, pregelaiinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a 51 WO 03/075839 568678 PCT/OS03/06451 ce]Mosic materia] (e.g.9 raicrocryBtalline ceDuiose), an acrylate (e.g„. polymefhylacrylaie), calcium carbonate, magnesium oxide, talc, or mixtures thereof For liquid formulations, pharmaceuticaliy acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oik. Examples of non-aqueous solvents 5 are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, inr-lnrKng saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solutions or suspensions can also 10 include the following components: a sterile diluent such -as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents fin: the 15 adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In addition, the compositions may further comprise binders (e.g.» acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyi cellulose, hydroxyprqpyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato 20 starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch giycolate, Primogel), buffers (e.g., tris-HCL, acetate, phosphate) of various pH -and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, soiubilizing 25 agents (e.g., glycerol, polyethylene glycerol), a glidant (e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, bulylated hydroxyanisole), stabilizers (e.g., hydroxypropyi cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, 30 methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g^ colloidal silicon dioxide), plasticizers (e.g., diethyl phfhalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyi cellulose, sodium 52 WO 03/075839 568678 PCT/US03/064S1 lauryl sulfate), polymer coatings (e.g., poloxamers ar polaxamioes), coating and film fanning agents (e.g, efhyl cellulose, acrylates, polymethacrylates) and/or adjuvants. In one embodiment, the active compounds are prepared with carriers that will protect fhe compound against rapid elimination fiom fhe body, such as a controlled release 5 formulation, including i™pknf-g and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, poiyglycolic acid, collagen, polyorfhoesters, and polylactic acid. Methods for preparation of such, formulations will be apparent to those skilled in the art The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. liposomal 10 suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceuticaliy acceptable earners. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral compositions in dosage unit form 15 for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each, unit containing a predetermined quantify of active compound calculated to produce fhe 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 20 dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for fee treatment of individuals. Ihe pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. 25 The daily administration is then repeated continuously for a period of several days to several years. Oral treatment may continue for "between one week and fhe life of the patient Preferably ihe administration takes place for five consecutive days after which time the patient can be evaluated to determine if further administration is required. The administration can be continuous or intermittent, i.e., treatment ibr a number of 30 consecutive days followed by a rest period. The compounds of the present invention maybe administered intravenously on the first day of treatment, with oral administration on fhe second day and all consecutive days thereafter. 53 WO 03/075839 568678 PCT/US03/06451 The compounds of the present invention may bs administered for fee purpose of preventing disease progression or stabilizing tumor growth. The preparation of pharmaceutical compositions that contain an active component is well understood in ihe ait, for example, by mixing, granulating, or tablet-forming 5 processes. The active therapeutic ingredient is often mixed with excipisnts that are pharmaceuticaliy acceptable and compatible with Ihe active ingredient For oral administration, the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert dilaants, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, 10 aqueous, alcoholic or oily solutions and Ihe like as detailed above. Hie compounds of the present invention may be administered at orally at a. total ".Taily dose of between 25 to 4000 mg/m2, for example, about 25 to 1000 mg, 50-1000 mg, 100 mg, 200 mg, 300 mg, 400 mg, 600 mg, 800 mg, 1000 mg and the like. Typically the compound is administered as a single dose when administering up to 400 mg to the 15 patient For higher total dosages (i.e., greater than 400 mg), the total is split into multiple dosages, for example, twice daily, three times daily or the like, preferably spread out over equal periods of time during the day. For example, two doses, e.g., 500 mg each, can be administered 12 hours apart to achieve a total dosage of 1000 mg in a day. In one currently preferred embodiment, SAHA or any of the HDAC inhibitors are 20 administered to the patient at a total daily dosage of 200 mg, In another currently preferred embodiment, SAHA or any of the HDAC inhibitors are administered to the patient at a total daily dosage of 400 mg. In another currently preferred embodiment, SAHA or any of the HDAC inhibitors are administered to the patient at a total daily dosage of 600 mg. 25 The amount of the compound administered to the patient is less than an amount that would cause toxicity in the patient In fhe certain embodiments, the amount of the compound that is administered to the patient is less than fhe amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of fee compound. Preferably, fhe concentration of the compound in the patient's plasma is 30 maintained at about 10 nM. In another embodiment, the concentration of fhe compound in fhe patient's plasma is maintained al about 25 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM. Ia another embodiment, the concentration of the compound in fee patient's plasma is maistained at about 100 nM. In another embodiment, the concentration of fhe compound 54 568678 WO 03/075839 PCT/US03/0M51 in the patient's plasma is maintained at about 500 nM. ia another embodiment, the concentration of fee compound in the patient's plasma is maintained at about 1000 nM. In another embodiment, the concentration of fee compound in fee patient's plasma is maintained at about 2500 nM. In another embodiment, fee concentration of Ihe compound 5 in fee patient's plasma is maintained at about 5000 nM. It has been, found with HMBA that administration of fee compound in an amount fiom about 5 gm/m2/day to about 30 gm/m2/day, particularly about 20 gm/m2/days is effective -without producing toxicity in fee patient The optimal amount of fhe compound that should be administered to fee patient in fee practice of the present invention will depend on the particular compound used and fee 10 type of cancer being treated In a currently preferred embodiment of fee present invention, fee pharmaceutical composition comprises a histone deacetylase (HDAC) inhibitor, microcrystalline cellulose as a carrier or diluent; croscarmellose sodium as a disintegrant; and magnesium stearate as a lubricant In a particularly preferred embodiment, fee HDAC inhibitor is suberoylanilide 15 hydroxamic acid (SAHA). The percentage of fee active ingredient and various excipients in fee formulations may vary. For example, fee composition may comprise between 20 and 90%, preferably between 50-70% by weight of fee histone deacetylase (HDAC). Furthermore, the composition may comprise between 10 and 70%, preferably between 20-40% by weight 20 microcrystalline cellulose as a carrier or diluent Furthermore, fee composition may comprise between 1 and 30%, preferably 5-15% by weight croscarmellose sodium as a disintegrant. Furthermore, fee composition may comprise between 0.1-5% by weight magnesium stearate as a lubricant. In another, preferred embodiment, fee composition comprises about 50-200 mg of fee HDAC inhibitor (e.g., 50 mg, 100 mg and 200 mg for 25 fee HDAC inhibitor, for example, SAHA). In a particularly preferred embodiment, fee composition is in the form of a gelatin c^jsule. A currently preferred embodiment of fee invention is a solid formulation of SAHA wife microcrystalline cellulose, NF (Avicel Ph 101), sodium croscarmellose, NF (AC-Di-Sol) end magnesium stearate, NF, contained in a gelatin capsule. A further prefixed 30 embodiment is 200 mg of solid SAHA wife 89.5 mg of microcrystalline cellulose, 9 mg of sodium croscarmellose and 1.5 mg of magnesium stearate contained in a gelatin capsule. It should be apparent to a person skilled in the art feat the pharmaceutical compositions of fee present invention are not only useful for inhibiting fee proliferation of neoplastic cells induction and treatment of cancer, and feat these compositions are useful 55 WO 03/075839 568678 PCT7US03/Q6451 in treating a wids range of diseases for which HDAC inhibitors have bssn found useful. For example, HDAC inhibitors, and hi particaiar SAHA, have been found to be useful in the treatment of a variety of acute and chronic inflammatory diseases, autoimmune diseases, allergic diseases, diseases associated with oxidative stress, and 5 diseases characterized by cellular hyperproliferation. Non-limiting examples are inflammatory conditions of a joint including and rheumatoid arthritis (RA) and psoriatic arthritis; inflammatory bowel diseases such as Crohn's disease and ulcerative colitis; spondyloarthropathies; scleroderma; psoriasis (including T-cell mediated psoriasis) and inflammatory dermatoses such an dermatitis, eczema, atopic dermatitis, allergic contact 10 dermatitis, urticaria; vasculitis (e.g., necrotizing, cutaneous, and hypersensitivity vasculitis); eosinphilic myositis, eosinophilic fasciitis; cancers with leukocyte infiltration of tie skin or organs, ischemic injury, including cerebral ischemia (e.g., biain injury as a result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to neurodegeneration); HTV, heart failure, chronic, acute or malignant liver disease, 15 autoimmune thyroiditis; systemic lupus erythematosus, Sjorgren's syndrome, lung diseases (e.g., ARDS); acule pancreatitis; amyotrophic lateral sclerosis (ALS); Alzheimer's disease; cachexia/anorexia; asthma; atherosclerosis; chronic fatigue syndrome, fever, diabetes (e.g., insulin diabetes or juvenile onset diabetes); glomerulonephritis; graft versus host rejection (e.g, in transplantation),; hemohonagic 20 . shock;,hyperalgesia: inflammatory bowel disease; multiple sclerosis; myopathies (e.g., nuiscle protein metabolism, esp. in sepsis); osteoporosis; Parkinson's disease; pain; preterm labor; psoriasis; reperfusion injury; cytokihe-mduced toxicity (e.g., septic shock, endotoxic shock); side effects from radiation therapy, temporal mandibular joint disease, tumor metastasis; or an inflammatory condition resulting fiom strain, sprain, cartilage 25 damage, trauma such as bum, orthopedic surgery, infection or other disease processes. Allergic diseases and conditions, include but are not limited to respiratory allergic diseases such as asthma, allergic rhinitis, hypersensitivity lung diseases, hypersensitivity pneumonitis, eosinophilic pneumonias (e.g., Loeffler's syndrome, chronic eosinophilic pneumonia), delayed-type hypersentitivity, interstitial lung diseases (ILD) (e.g., idiopathic 30 pulmonary fibrosis, or ED associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclesrosis, Sjogren's syndrome, polymyositis or dennalomyositis); systemic anaphylaxis or hypersensitivity responses, drug allergies (e.g., to penicillin, cephalosporins), insect sting allergies, and the like. For example, HDAC inhibitors, and in particaiar SABA, have been found to be 56 568678 WO 03/075839 PCT/nS03/f)6451 useful in the treatment of a variety of neurodegenerative diseases, a noa-exhaustive list of which, is: L Disorders characterized by progressive dementia in the absence of other prominent 5 neurologic signs, such as Alzheimer's disease; Senile dementia of the Alzheimer type; and Pick's disease (lobar atrophy). IL Syndromes combining progressive dementia with other prominent neurologic abnormalities such as A) syndromes appearing mainly in adults (e.g., Huntington's 10 disease, Multiple system atrophy combining dementia with ataxia and/onnamfestations of Parkinson's disease, Progressive supranuclear palsy (Steel-Richardson-Olszewski), xiifitise. Lewy body disease, and corticodentatorrigral degeneration); and B) syndromes appearing mainly in children or young adults (e.g., Hailervorden-Spaiz disease aad progressive fiwwiKnl myoclonic epilepsy). 15 TTT Syndromes of gradually developing abnormalities of posture and movement such as paralysis agitans (Parkinson's disease), striatonigral degeneration, progressive supranuclear palsy, torsion dystonia (torsion spasm; dystonia musculorum deformans), spasmodic torticollis and other dyskmesis, familial tremor, and Gilles de la Touretfe 20 syndrome. IV. Syndromes of progressive ataxia such as cerebellar degenerations (e.g., cerebellar cortical degeneration and olivopontocerebellar atrophy (OPCA)); and spinocerebellar degeneration (Friedreich's alazia and related disorders). 25 V. Syndrome of central autonomic nervous system failure (Shy-Drager syndrome). VI Syndromes of muscular weakness and wasting without sensory changes (motomeuron disease such as amyotrophic lateral sclerosis, spinal muscular atrophy (e.g., 30 infantile spinal muscular atrophy (Werdaig-Hoffinan), juvenile spinal muscular atrophy (Wohlfart-Kagelberg-Welander) and other forms of familial spinal muscular atrophy), primary lateral sclerosis, and hereditary spastic paraplegia. VII. Syndromes combining muscular weakness and wasting with sensory changes 57 WO 03/075839 568678 PCT/USC3/06451 (progressive neural muscular atrophy, chronic familial polyneuropathies) such as peroneal muscular atrophy (Charcot-Marie-Tooth), B. Hypertrophic interstitial polyneuropathy (Dejerine-Sottas), and C. Miscellaneous forms of chronic progressive neuropathy, 5 VIE Syndromes of progressive visual loss such as pigmentary degeneration of the retina (retinitis pigmentosa), and hereditary optic atrophy (Leber's disease). The invention is illustrated in fee examples in the Experimental Details Section ■which follows. This section is set forth to aid in an understanding of the invention but is 10 not intended to, and should not be constmed to limit in any way the inventioai as set forth in the claims which follow thereafter. 58 WO 83/075839 568678 PCT/DS03/064S1 EXPERIMENTAL DETAILS SECTION EXAMPLE 1: Synthesis of SAHA SAHA can be synthesized according to the method outlined below, or according to the method set forth in US Patent 5,369,108, the contents of which are incorporated by reference in their entirety, or according to any other method. ■Synthesis of SABA -'Step 1 - Synthesis of-Subetaniiih add 0 0 jf-\ f {? f In a 22 L flask was placed 3,500 g (20.09 moles) of suberic acid, and the acid 10 melted with heat The temperature was raised to 175°C, and then 2,040 g (21.92 moles) of aniline was added. The temperature was raised to 190°C and held at that temperature for 20 minutes. The melt was poured into a Nalgeae tank that contained 4,017 g of potassium hydroxide dissolved in 50 L of water. The mixture was stirred for 20 minutes following the addition of the melt The reaction was repeated at the same scale, and the second melt 15 was 'poured into the same solution of potassium hydroxide. After the mixture was thoroughly stirred, the stirrer was tamed of£ and the mixture was allowed to settle. The mixture was then filtered through a pad of Celite (4,200 g) (the product was filtered to remove the neutral by-product (from attack by aniline on both aids of suberic acid). The filtrate contained the salt of the product, and also the salt of unreacted suberic acid. The 20 mixture was allowed to settle because fhe filtration was very slow, talcing several days.). The filtrate was acidified using 5 L of concentrated hydrochloric acid; the mixture was stirred for one hour, and then allowed to settle overnight. The product was collected by filtration, and washed on the funnel with deionized water (4x5 L). TTie wet filter cake was placed in a 72 L flask with 44 L of deionized water, the mixture heated to 50°C, and 25 fee solid isolated by a hot filtration (the desired product was contaminated with suberic acid which is has a much greater solubility in hot water. Several hot triturations were done 59 568678 WO 03/075839 PCT/US03/06451 to remove suberic acid. The product was checked by NMR. [DgDMSO] to monitor fee removal of suberic acid). Hie hot trituration was repeated -with 44 L of water at 50°C. The product was again isolated by filtration, and rinsed -with 4 L of hot ■water, it was dried over (he weekend in a vacuum oven at 65°C using a Nash pump as fhe vacuum source (the 5 Nash pump is a liquid ring pump (water) and pulls a vacuum of about 29 inch of mercury. An intermittent argon purge was used to help carry off water); 4,182.8 g of suberanilic add was obtained. The product still contained a small amount of suberic acid; therefore the hot trituration was done portionwise al 65°C, using about 300 g of product at a time. Each 10 portion was filtered, and rinsed thoroughly with additional hot water (a total of about 6 L). This was-repeated to purify the entire .batch. This completely removed suberic acid from fee product The solid product was combined in a flask and stirred with 6 L of methanol/water (1:2), and then isolated by filtration and air dried on lie filter over the week end. It was placed in trays and dried in a vacuum oven at 65°C for 45 hours using 15 fhe Nash punp and an argon bleed. The final product has a weight of 3,278.4 g (32.7% yield). 60 WO 03/075839 568678 PCT/USC3/06451 stsgjiZ- ■4t-*c—'(cs^—d—qcsfc F r -^C^(CH2)f-C-0H To a. 50 L flask fitted wifh a mechanical stirrer, and condenser was placed 3,229 g of suberanilic acid from the previous step, 20 L of methanol, and 398.7 g of Dowex 5OWX2-40O resin. The mixture, was heated to refine and held at reflux for 18 hours. He • mixture was filtered to remove fhe resin beads, and the filtrate was taken to a residue on a xotary evaporator. The residue from the rotary evaporator was transferred into a 50 L fiask fitted wifh a condenser and mechanical stirrer. To fhe flask was added 6 L of methanol, and the mixture heated to give a solution. Thai 2 L of deionized water was added, and the heat tamed ofE The stirred mixture was allowed to cool, and then the flask was placed in an ice hatbj and the mixture cooled. Ihe solid product was isolated hy filtration, and the filter cake was rinsed with 4 L of cold methanol/water (1:1). The product was dried at 45°C in a vacuum oven using a Nashpomp for a total of 64 hoars to give 2,850.2 g (84% yield) of mefhyl suberanilate, CSL Lot # 98-794-92-3 1. - afQtt'fe.SAgA To a 50 L flask with a mechanical stirrer, thermocouple, and inlet for inert atmosphere was added 1,451.9 g of hydroxylamine hydrochloride, 19 L of anhydrous methanol, sod a 3.93 L of a 30% sodium methoxide solution in methanol. The flaalr was then charged with 2,748.0 g of methyl suberanilate, followed by 1.9 L of a 30% sodium mefhoxide solution in methanol Ths mixture was allowed to stir for 16hr aad 10 minutes. Approximately one half of the reaction mixture was transferred from fhe reaction (flask 1) to & 50 L flask (flask 2) fitted wifh a mechanical stirrer. Thai 27 L of deionized water was added to flask 1 and fhe mixture was stirrer for 10 minutes. The pH was taken using a pH meter; the pH was 11.56. Ihe pH of fhe mixture was adjnsted to 12.02 by fhe 61 WO 03/075839 PCT/US03/06451 addition of 100 ml of fee 30% sodium methoxide solution is methanol; this gave a clear solution (tie reaction mixture at this time contained a small amount of solid. The pH was adjusted to give a clear solution from which the precipitation the product would be precipitated). The reaction mixture in flask 2 was diluted in the same maimer; 27 L of deionized water was added, and the pH adjusted by the addition of 100 ml of a 30 % sodium methoxide solution to the mixture, Id give a pH of 12.01 (clear solution). The reaction mixture in each flask was acidified by the addition of glacial acetic acid to precipitate the product. Flask 1 had a final pH of 8.98, and flask 2 bad a final pH of 8.70. The product fiom both flasks was isolated by filtration using a Buchner funnel and filter cloth. The filter cake was washed with 15 L of deionized water, and fhe fiomel was .covered and the product was partially dried on the funnel under vacuum for 15.5 hr. The product was removed and placed into five glass trays. The trays were placed in a vacuum oven and the product was dried to constant weight. The first drying period was for 22 hours at 60DC "g"»g a Nash pump as the vacuum source with an argon bleed. The trays were removed from the vacuum oven and weighed. The trays ware returned to the oven and the product dried for an additions} 4 hr and 10 minutes using an oil pump as the • vacuum source and with no argon bleed. The material was packaged in double 4-miIl polyethylene bags, and placed in a plastic outer container. The final weight after sampling was 2633.4 g (95.6%). Step 4 - Recrystolfeation of Crude SAHA The crude SAHA was recrystallized from methanol/water. A 50 L flask with a mechanical stirrer, thermocouple, condenser, and inlet for inert atmosphere was charged wifh the crude SAHA to be crystallized (2,525.7 g), followed by 2,625 ml of deionized water and 15,755 ml of methanol. The material was heated to reflux to give a solution. Then 5,250 ml of deionized water was added to the reaction mixture. The heat was tamed of£ and the mixture was allowed to cooL When fhe mixture had cooled sufficiently so that the flasV could be safely handled (28°C), the flask was removed from the heating mantle, and placed in a tub far use as a cooling bath. Ice/water was added to the tub to cool the mixture to -5°C. The mixture was held below that temperature for 2 hours. The product was isolated by filtration, and the filter cake washed with 1.5 L of cold methanol/water (2:1). The funnel was covered, and the product was partially dried under vacuum for 1.75 hr. The product was removed from fhe fennel and placed in 6 glass trays. The bays were placed in a vacuum oven, and the product was dried for 64.75 hr at 60°C using a Nash 568678 WO 03/075839 PCT/CS03/06451 pump as fhe vacuum source, aad using an argon bleed. The trays were removed fox weighing, and then returned to the oven and dried for an additional 4 hours at 60aC to give a constant weight The vacuum source for the second drying period was a oil pump, and no argon bleed was used. The material was packaged in double 4-mill polyethylene bags, and placed in a plastic outer container. The final weight after sampling was 2,540S g (92.5%;. EXAMPLE 2: Oral dosing O'fphFanylairilidehvdrOYnm^ arid fKATTA^ I Background: Treatment with hybrid polar cellular differentiation agents has resulted in the inhibition of growth oflhuman solid tumor derived cell lines and xenografts. The effect is mediated in part by inhibition of histone deacetylase. SAHA is a potent histone deacetylase inhibitor that has been shown to have the ability to induce tumor cell growth airest, differentiation aad apoptosis in the laboratory and in preclinical studies. Objectives: To define a safe daily oral regimen of SAHA that can be used in Phase II studies. In addition, the pharmacokinetic profile of the oral formulation of SAHA was be evaluated. The oral bioavailability of SAHA in humans in the fasting vs; non-fasting state and antitumor effects of treatment were also monitored. Additionally, the biological effects of SAHA on normal tissues and tumor cells were assessed and responses with respect to levels of histone acetylation were documented. Patients: Patients with histologically documented advanced stage, primary or metastatic adult solid tumors that are refractory to standard therapy or for which no curative standard therapy exists. Patients must have a Kamofsky Performance Status of £70%, and adequate hematologic, hepatic and renal function. Patients must be at least four weeks fiom any prior chemotherapy, radiation therapy or other investigational anticancer drugs. Dosing Schedule: On the first day, patients were first treated with 200 mg of intravenously-administered SAHA. Starting on the second day, patients were treated wifh daily doses of oral SAHA according to Table 1. Each cohort received a different dose of SAHA. "QD" indicates dosing once a day; "Q12 hours" indicates dosing twice a day. For example, patients in Cohort IV received two 800 mg doses of SAHA per day. Doses were administered to patients daily and continuously. Blood samples were taken on day one and on day 21 of oral treatment Patients were taken off oral SAHA treatment due to 63 568678 WO 03/075839 PCT/US03/06451 disease progression, tumor regression, unacceptable side effects, or treatment with other therapies. Table 1: Oral SAHA Dose Schedule Cohort Oral Dose (mg) Number of Days Daily Dosing Schedule I 200 Continuous QD n 400 Continuous QD m 400 Continuous Q12 hours IV 800 Continuous Q12 hours V 1200 Continuous Q12 hours VI 1600 Continuous -Q12hours vn 2000 Continuous Q12 hours •Results: Comparison of serum plasma levels shows high bioavailability of SAHA administered orally, both when fee patient fasted and when the patient did not fast, compared to SAHA administered intravenously (TV SAHA). "AUC" is an estimate offhe bioavailability of SAHA in (ng/ml)min, where 660 ng/iml is equal to 2.5 (jM SAHA Hie AUC taken together with the half-life (t^) shows that the overall bioavailability of oral SAHA is better than that of IV SAHA. Cna* is the maximum concentration of SAHA observed after administration. IV SAHA was administered at 200 mg infused over two hours. The oral SAHA was administered in a single capsule at 200 mg. Tables 2 and 3 summarize the results of an HPLC assay (LCMS using a deuterated standard) that quantitates the amount of SAHA in the blood plasma of Ihe patients versus time, using acetylated histone-4 (ct-AcH4) as a marker. Table 2: Serum Plasma Levels of Oral SAHA - Patient #1 IV Oral (fasting) Oral (nonfasting) Cmax (ng/ml) 1329 225 328 tvi (min) 20 80 64 AUC (ng/ml)mm 153,000 25,000 59,000 Table 3: Serum Plasma Levels of Oral SAHA - Patient #2 > IV Oral (fasting) Oral (nonfasting) 64 WO 03/075839 568678 PCT/PSQ3/06451 Cniax (UgAlll) 1003 362 302 tisCmin) 21 82 93 AUC (ng/ml)min 108,130 63,114 59,874 Figures 1 to 8 are HPLC slides showing the amount of a-Ac54 in patients in Cohorts I and H, measured ai up to 10 hours after receiving the oral dose, compared with the a-AcH4 levels when SAHA was administered intravenously. Fig 9 shows ths mean plasma concentration of SAHA (ng/ml) at the indicated time points following administration. Fig 9A: Oral dose (200 mg and 400 mg) under tasting on Day 8. Fig 9B: Oral dose with food-on Day-9. Fig9C: IV dose on day 1. Fig 40 shows-the apparent half-life of a SAHA 200 mg and 400 mg oral dose, on Days 8, 9 and 22. Fig 11 shows fhe AUC (ng/ml/hr) of a SAHA 200 mg and 400 mg oral dose, on Days 8,9 and 22. Figure 12 shows fhe bioavailability of SAHA after a 200 mg and 400 mg oral dose, on Days 8, 9 and 22. EXAMPLE 3: Oral dnsinp nf suberoylanilide hvdTrrevamin acid fSAWAt - Dose Escalation. In another experiment, twenty-five patients wifh solid tumors have been enrolled onto arm A, thirteen patients with Hodgkin's or non-Hodgkm's lymphomas have been enrolled onto arm B, and one patient with acute leukemia and one patient with myelodysplastic syndrome have been enrolled onto arm C, as shown in Table 4. Table 4: Dose Escalation Scheme and Number of Patients on Each Dose Level Cohort Dose (mg/day) Dosing Schedule #Days of Dosing Rest Period #Patients Enrolled (aim A/ann B/aim C)* r 200 Once a day Continuous None 6/0/0 n 400 Once a day Caatmuaus None 5/4/2 m 400 q 12 boors Continuous None 6/3/0 IV 600 Once a day Continuous None 4/3/0 V 200 q 12 boras Continuous None 4/3/0 VI 300 q 12 hoars Continuous None -/-/- Sub-totals: 25/13/2 Total = 40 ♦Aim A= solid tumor, ami B= iymphomE, aim C= leutomi* 65 568678 WO 03/075839 PCT/US03/06451 Results: Among eleven patients treated in Cohort H, one patient experienced the DLT of grade 3 diarrhea and grade 3 dehydration daring the first treatment cycle. Nine patients were entered into Cohort HI. Two patients were inevatuable for the 28-day toxicity stspfiggmgnf- because of early study termination due to rapid progression of disease. Of the seven remaining patients, five experienced DLT during the first treatment cycle: diarnhea/dehydration (n=l), fatigue/dehydration (n=l), anorexia (n=l), dehydration (n=l) and anorexia/dehydration (n=l). These five patients recovered in approximately one week after the study drug was held. They were subsequently dose reduced to 400 mg QD which, appeared to be well tolerated. The median days on 400 mg BID for all patients in Cohort m was 21 days. Based on these findings the 400 mg ql2 hour dosing schedule was judged to have exceeded fhe maximally tolerated dose. Following protocol amendment, accrual was continued in cohort IV at a dose of600 mg once a day. Of fee seven patients enrolled onto cohort IV, two were inevaluable for fhe 28-day toxicity assessment because of early stady temrrnatinn due to rapid progression of disease. Three patients experienced DLT during the first treatment cycle: anorexia/dehydration/faiigue (n=l), and diarrhea/dehydration (n=2). The 600 mg dose was therefore judged to have exceeded the maximally tolerated dose and the 400 mg once a day dose was defined as fhe maximally tolerated dose for once daily oral administration. The protocol was amended to evaluate pHftitinnal dose levels of the twice a day dosing schedule at 200 mg BID and 300 mg BID administered continuously. The interim pharmacokinetic analysis was based on 18 patients treated on the dose levels of 200 mg QD, 400 mg QD, and 400 mg BID. In general, fhe mean estimates of Cnra and AUCfof of SAHA administered orally under fasting condition or with food tn creased proportionally with dose in fhe 200 mg to 400 mg dose range. Overall, the fraction of AUCj„f due to extrapolation was 1% or less. Mean estimates for apparent half-life were variable across dose groups under fasting condition or with food, ranging from 61 to 114 minutes. The mean estimates of Cmax, varies fiom 233 ng/ml (0.88 pM) to 570 ngfail (2.3 pM). The bioavailable fraction of SAHA, calculated fiom the AUChj values after the IV infusion and oral routes, was found to be approximately 0.48. Peripheral blood mononuclear cells were collected pre-therapy, immediately post-infiision and between 2-10 hours after oral ingestion of the SAHA capsules to assess the 66 WO 03/075839 568678 PCT/US03/0&451 effect of SAHA on Ihs extent of histone acetylation in & normal host celL Histanes were isolated aad probed with anti-acetylated histone (H3) antibody followed by HRP-secondary antibody. Preliminary analysis demonstrated an increase in the accumulation of acetylated histones in peripheral mononuclear cells that could be detected up to 10 hours after ingestion of SAHA capsules at 400 mg per day dose level. Thirteen patients continued treatment for 3-12 months with responding or stable disease: thyroid (n=3), sweat gland (n=l), renal (n=2)„ larynx (n=l), prostate (n=l), Hodgkin's lymphoma (n=2), ncm-Hodgkin's lymphoma (n=2). and leukemia (n=I). Six patients had tumor shrinkage on. CT scans. Three of these six patients meet the criteria of partial response (one patient with metastatic laryngeal cancer and two patients with non-Hpdgkin's lymphomas). These partial responses occurred at the dose levels of 400 mg BE) (n=2) and 600 mg QD (u=l). EXAMPLE 4: Intravenous Dosing of SAHA Table 5 shows a dosing schedule for patients receiving SAHA intravenously. Patients begin in Cohort I, receiving 300 mg/m2 of SAHA for five consecutive days in a week for one week, for a total dose of 1500 mg/m2. Patients were then observed for a period of two weeks and continued to Cohort H, then progressed through the Cohorts unless treatment was terminated due to disease progression, tumor regression, unacceptable side effects or the patient received other treatment Table 5: Standard Dose Escalation for Intravenously-Administered SAHA Cohort Dose (mg/m2) Numbs- of Days/Week Number of Consecutive Weeks Observation Period (Weeks) Total Dose (mg/m2) I 300 5 1 2 1500 n 300 5 2 2 3000 m 300 5 3 1* 4500 IV 600 5 3 1* 9000 V 800 5 3 1* 23500 VI 1200 5 3 1* 18000 vn 1500 5 3 1* 22500 ♦Hematologic patients started at dose level HI. 67 568678 WO C3/P75£2S PCT/OSI&/D$4£l The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that thai prior art forms part of the common general knowledge in New Zealand. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say. in the sense of "including, but not limited to". While feis irvemion b2S been pariiciilsdy shown and described wife references to preferred embodiments thereof it win be understood by these skilled in fee art fKaf vsrians changes in form and details msy be made therein without departing from fhe mpmmg 0f the iuveiihon described. Rather, fee scope of fhe inveniian is defined by ftte claims fhgf follow: 6S 568678 WO 03/075839 PCT/CS03/06451 References 1. Sporn, M. B., Robots, A B., andPriscoll, J. S. (1985) in Cancan Principles and Practice of Oncology, eds. Hellman,, S, Rosenberg, S. A, and DeVita, V. T., Jr., Ed. 2, (J. B. Lippincott, Philadelphia.), P. 49. 2. Bieitman, T. R., Selonicfc, S. E., and Collins, S. I. (1980) Proc. Natl. Acad. Sri. USA 77:2936-2940. 3. Olsson, IL andBreitman, T. R. (1982) Cancer Res. 42:3924-3927. 4. Schwartz, E. L and Sartorelli, A. C. (1982) Cancer Res. 42: 2651-2655. 5. Marks, P. A_, Sheffery, ML, and Rifkind, R. A. (1987) Cancer Res. 47:659. 6. Sachs, L. (1978J Natare (Lond.) 274: 535. 7. Friend, C., Scher, W., Holland, J. W., and Sato, T. (1971) Proc. NaflL Acad. ScL (USA) 68:378-382. 8. Taaaka, M., Levy, J., Tarada, M., Breslow, R., Rifkind, R, A., and Marks, P, A. (1975) Proc. Nail Acad. Sci. (USA) 72:1003-1006. 9. Reuben, R. C-, Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976) Proc. NatL Acad. ScL (USA) 73: 862-866. 10. Abe, E., Miyanra, C., Sakagami", EL, Takeda, M, Konno, EL, YamazaTri, T., Yoshika, S., and Suda, T. (1981) Proc. Natl, Acad, ScL (USA) 78:4990-4994. 11. Schwartz, E. L., Snoddy, J. R., Krentter, D., Rasmussen, H., and Sartorelli, A. C. (1983) Proc. Am. Assoc. Cancer Res. 24:18. 12. Tanenaga, K., Hozumi, M., and Sakagami, Y. (1980) Cancer Res. 40:914-919. 13. Lotem, J. and Sachs, L. (1975) hat J. Cancer 15:731-740. 14. Metcalt D. (1985) Science, 229:16-22. 15. Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. HematoL 11:490-498. 16. Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. & Biophys. Res. Comm. 109: 348-354. 17. Hubeiman, E. and Callaham, M. F. (1979) Proc. Natl, Acad. Sci. (USA) 76:1293-1297. 18. Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sci. (USA) 76: 5158-5162. 19. Terada, M., Epner, E., NudeL, U, Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978) Proc. NafL Acad. Sci. (USA) 75:2795-2799. 69 WO 03/075839 568678 PCT/CS03/06451 20. Morin, M. J. and Sartorelli, A C. (1984) Cancer Rea. 44:2807-2812. 21. Schwartz, E. L, Brown, B. I., Nierenberg, M., Maish, J. C., and Sartorelli, A. C. (1983) Cancer Res. 43:2725-2730. 22. Sugano, EL, Furusawa, M., Kawagudhi, T., and Dcawa, Y. (1973) BibL HsraatoL 39: 943-954. 23. Ebert, P. S., Wars, L, andBuell, D. N. (1976) Cancer Res. 36:1809-1813. 24. Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70:235-238. 25. Fibach, E., Reuben, R. C., Rifkind, R. A., and Marks, P. A. (1977) Cancer Res. 37: 440-444. 26. Melloni, E., Pontremoli, S., Damiani, G., Viotti, P., Weich, N., Rifkind, R. A., and Marks, P. A (1988) Proc. Natl. Acad. Sci. (USA) 85:3835-3839. 27. Reuben, R., Khanna, P. L., Gazitt, Y., Breslow, R., Rifkind, R A, and Marks, P. A (1978) J. Biol. Chem. 253:4214-4218. 28. Marks, P. A. and Rifldnd, R. A (1988) International Journal of Cell Cloning 6:230-240. 29. Melloni, E., Pontremoli, S., Mlchetti, M., Sacco, O., Cakirogin, A G., Jackson, J. F., Rifkind, R. A., and Maries, P. A* (1987) Proc. Natl. Acad. Sciences (USA) 84:5282-5286. 30. Maries, P. A and Rifldnd, R. A (1984) Cancer 54:2766-2769. 31. Egorin, M. J., Sigman, L. M. VanEcho, D. A., Forrest, A., WMtacre, M. Y., and Aisner, J. (1987) Cancer. Res. 47:617-623. 32. Rowinsky, E. W., Ettinger, D. S., Grochow, L. B., Brundrett, R B., Cates, A E., and Donehower, R. C. (1986) J. Clin. CtacoL 4:1835-1844. 33. Rowinsky, E. L. Ettinger, D. S., McGmre, W. P., Noe, D. A, Grochow, L. B., and Donehower, R. C. (1987) Cancer Res. 47: 5788-5795. 34. Callery, P. S., Egorin, M. J., Geelhaar, L. A, and Nayer, M. S. B. (1986) Cancer Rss, 46:4900-4903. 35. Yotmg, C. W. Banucchi, M. P., Walsh, T. B., Blatzer, L., Yaldaie, S., Stevens, Y. W., Gordon, C., Tang, W., RLBrind, R. A, and Marks, P. A. (1988) Cancer Res. 48: 7304-7309. 36. Andreefij M., Young, C., Clarkson, B., Fetten, J., Rifkind, R. A, and Marks, P. A. (1988) Blood 72:186a. 70 568678 WO 03/075839 PCT/DS03/06451 37. Marks, P. A., Breslow, R., Rifkind, R A, Ngo, L., and Singh, R. (19S9) Proc. NafL Acad. ScL (USA) 86: 6358-6362. 38. Breslow, R., Jursic, B., Yan, Z. F., Friedman, E., Leng, L., Ngo, L., Rifkind, EL A., and Marks, P. A. (1991) Proc. Natl. Acad. Sri. (USA) 88: 5542-5546. 39. Richon, V.M., Webb, Y., Msrgar, R., et aL (1996) PNAS 93:5705-8. 40. Cohen, L.A, Aroin, S., Marks, PA., Rifkind, RA-, Desai, D., and Richon, VM. (1999) Anticancer Research 19:4999-5006. 41. Grunstein, M. (1997) Nature 389:349-52. 42. Frnnm, M.S., Donigjan, JR., Cohen, A., et aL (1999) Nature 401:188-193. 43. Van Lint, C., Tfofliatil, S., Verdin, E. (1996) Gene Expression 5:245-53. 44. Archer, S. Shufen, M. Shei, A, Hodin, R. (1998) PNAS 95:6791-96. 45. Dressel, U., Renkawitz, R., Baniahmad, A (2000) Anticancer Research 20(2A):1017-22. 46. Lin, RJ., Nagy, L., Inoue, S., et aL (1998) Natore 391:811-14. 71 568678 WO 03/075839 PCT/US03/06451 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> What is claimed is: 1. An active ingredient consisting of a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtained by a method 5 comprising the step of recrystailizing a crude preparation of SAHA from a mixture of methanol and water. 2. The active ingredient of claim 1, wherein the mixture of methanol and water is a mixture of about 2:1 of methanol/water. 10 3. An active ingredient consisting of a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtained by a method comprising the steps of: a. reacting suberic acid with aniline to form suberanilic acid having the structure: or a salt thereof; b. reacting the suberanilic acid or the salt thereof with methanol to form methyl suberanilate having the structure: 20 c. reacting the methyl suberanilate with hydroxylamine hydrochloride to form a crude suberoylanilide hydroxamic acid in a reaction mixture; and d. recrystailizing a crude preparation of SAHA from a mixture of methanol and water. 301227341 l.DOC 72 INTELLECTUAL PROPERTY OFFICE OF N.Z. -t SEP 2009 RECEIVED 568678 WO 03/075839 PCT/US03/06451 4. The active ingredient of claim 3, wherein the mixture of methanol and water is a mixture of about 2:1 of methanol/water. 5. An active ingredient as claimed in claim 1 or 3 substantially as herein described 5 with reference to any one of the examples and/or the accompanying Figures. 7. A pharmaceutical composition comprising the active ingredient of any one of claims 1-6 and a pharmaceuticaliy acceptable carrier, wherein the composition is formulated as a solid dosage form. 15 8. A pharmaceutical composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtained by a method comprising the step of recrystailizing a crude preparation of SAHA from a mixture of methanol and water, and a pharmaceuticaliy acceptable carrier, wherein the composition is 20 formulated as a solid dosage form. 9. The pharmaceutical composition of claim 8, wherein the mixture of methanol and water is a mixture of about 2:1 of methanol/water. 25 10. A pharmaceutical composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtained by a method comprising the steps of: a. reacting suberic acid with aniline to form suberanilic acid having the structure: 6. The active ingredient of claim 3, wherein step (c) further comprises the steps of: (1) adding sodium methoxide to the reaction mixture to obtain a clear solution; and (2) adding glacial acetic acid to the clear solution to form a precipitate comprising 10 crude suberoylanilide hydroxamic acid. h o oh o 30 or a salt thereof; 301227341 l.DOC intellectual property off ice of m.z 73 -'t SEP 2009 RECEIVED 568678 WO 03/075839 PCT/US03/06451 b. reacting the suberanilic acid or the salt thereof with methanol to form methyl suberanilate having the structure: c. reacting the methyl suberanilate with hydroxylamine hydrochloride to form a crude suberoylanilide hydroxamic acid in a reaction mixture; and d. recrystailizing a crude preparation of SAHA from a mixture of methanol and water; and a pharmaceuticaliy acceptable carrier, 10 wherein the composition is formulated as a solid dosage form. 11. The pharmaceutical composition of claim 8, wherein the mixture of methanol and water is a mixture of about 2:1 of methanol/water. 15 20 12. The pharmaceutical composition of claim 10, wherein step (c) further comprises the steps of: (1) adding sodium methoxide to the reaction mixture to obtain a clear solution; and (2) adding glacial acetic acid to the clear solution to form a precipitate comprising crude suberoylanilide hydroxamic acid. 13. A pharmaceutical composition as claimed in any one of claims 7, 8 or 10 substantially as herein described with reference to any one of the examples and/or the accompanying Figures. 25 14. The pharmaceutical composition of any one of claims 7-13, that comprises about 50-200 mg of SAHA. 15. The pharmaceutical composition of any one of claims 7-13, that comprises about 100 mg of SAHA. 301227341 1.DOC 74 INTELLECTUAL PROPERTY OFFICE OF N.Z -4 SEP 2009 RECEIVED 568678 WO 03/075839 PCT/US03/06451 16. The pharmaceutical composition of any one of claims 7-13, that comprises about 400 mg of SAHA. 17. The pharmaceutical composition of any one of claims 7-13, that comprises about 5 200 mg of SAHA. 18. The pharmaceutical composition of any one of claims 7-13, that comprises SAHA; and one or more of microcrystalline cellulose; croscarmellose sodium; and magnesium strearate. 10 19. The pharmaceutical composition of any one of claims 7-13, that comprises up to about 400 mg of SAHA. 20. A composition comprising a crystalline form of suberoylanilide hydroxamic acid 15 (SAHA) having physical characteristics of crystalline SAHA obtained by a method comprising the step of recrystailizing a crude preparation of SAHA from a mixture of methanol and water. 21. The composition of claim 20, wherein the mixture of methanol and water is a mixture of about 2:1 of methanol/water. 20 22. A composition comprising a crystalline form of suberoylanilide hydroxamic acid (SAHA) having physical characteristics of crystalline SAHA obtained by a method comprising the steps of: a. reacting suberic acid with aniline to form suberanilic acid having the structure: or a salt thereof; b. reacting the suberanilic acid or the salt thereof with methanol to form methyl suberanilate having the structure: 301227341_1.DOC 75 INTELLECTUAL PROPERTY OFFICE OF N.Z SEP 2009 REC EIV ED 568678 c. reacting the methyl suberanilate with hydroxylamine hydrochloride to form a crude suberoylanilide hydroxamic acid in a reaction mixture; and d. recrystailizing a crude preparation of SAHA from a mixture of methanol and 5 water. 23. The composition of claim 22, wherein the mixture of methanol and water is a mixture of about 2:1 of methanol/water. 10 24. The composition of claim 22, wherein step (c) further comprises the steps of: (1) adding sodium methoxide to the reaction mixture to obtain a clear solution; and (2) adding glacial acetic acid to the clear solution to form a precipitate comprising crude suberoylanilide hydroxamic acid. IS 25. A composition as claimed in claims 20 or 22 substantially as herein described with reference to any one of the examples and/or the accompanying Figures. 20 SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH AND MERCK HDAC RESEARCH, LLC By their Attorneys BALDWINS 25 </div>