WO2011103563A1 - Methods and compositions for inhibiting and preventing the growth of malignant mast cells - Google Patents

Abstract

Methods and compositions for inhibiting or preventing the growth of malignant mast cells in an animal including administering a therapeutically effective amount of a histone deacetylase inhibitor to an animal to inhibit or prevent the growth of the malignant mast cells are provided.

Description

METHODS AND COMPOSITIONS FOR INHIBITING AND PREVENTING THE

GROWTH OF MALIGNANT MAST CELLS

FIELD

[0001] The present disclosure relates to the use of histone deacetylase inhibitors to treat cancerous cells and to methods of using histone deacetylase inhibitors to treat malignant mast cells.

BACKGROUND

[0002] Global DNA hypermethylation and histone hypoacetylation are hallmarks of many cancers. These epigenetic modifications alter gene expression in the absence of changes to DNA sequence and play important roles in tumorigenesis by modulating the expression of tumor suppressors, cell cycle regulatory and DNA repair genes. The potential reversibility of these epigenetic changes has made the related regulatory pathways attractive targets for therapeutic intervention. Histone deacetylase (HDAC) inhibitors are a promising class of anti-tumor agents that can induce growth arrest, differentiation and apoptosis of cancer cells through the

accumulation of acetylated histones leading to chromatin remodeling and the restored

transcription of genes regulating proliferation, cell cycle progression and cell survival.

[0003] In one aspect, HDACi is believed to alter the transcription of several genes such as p21 via histone modification. However, a growing number of non-histone substrates have been identified and implicated in the anti-tumor activities of HDAC inhibitors, including molecular chaperones, such as heat shock protein 90 (HSP90), and transcription factors, including STAT3 and NF- κΒ. Specifically, HSP90 is a substrate of HDAC6 and is hyperacetylated after HDAC inhibitor treatment resulting in the loss of chaperone function. This HSP90-dependent pathway has been recognized as an important histone acetylation-independent anti-cancer mechanism for the HDAC inhibitor-induced downregulation of Kit in human CML gastrointestinal stromal tumor cell lines, Bcr-Abl in human CML lines, estrogen receptor, and DNA methyltransferase 1 (DNMT1). [0004] Mast cell-associated malignancies are important diseases in both humans and dogs, and are characterized by activating mutations in Kit in a significant portion of patients. Over 90% of human patients with systemic mastocytosis (SM) carry the V816D mutation Kit and exhibit resistance to imatinib (Gleevec) therapy. Similarly, up to 30% of dogs with high grade mast cell tumors (MCT) possess internal tandem duplications (ITD) in the Kit juxtamembrane (JM) domain. Targeted inhibitors of Kit such as imatinib mesylate (GLEEVEC®) and toceranib phosphate (PALLADIA™) have demonstrated clinical efficacy against malignant mast cell disease. However, different Kit mutations exhibit variable resistance toward Kit inhibitors, and the potential development of secondary resistance mutations is a concern.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 (a) shows cell proliferation for P815, C2, and BR cells treated with increasing concentrations of AR-42 for 24 hours;

[0006] FIG. 1 (b) shows the level of cellular proliferation for P815, C2, and BR cells treated with 0.5 μΜ AR-42 for 24 hours;

[0007] FIG. 1(c) shows the percentage of apoptosis of P815, C2, and BR cells induced in cells treated with increasing concentrations of AR-42 and 1 μΜ 17-AAG for 24 hours;

[0008] FIG. 1 (d) shows caspase 3/7 activation in P815, C2, and BR cells incubated with various concentrations of AR-42 and 17-AAG for 24 hours;

[0009] FIG. 1(e) is a western blot showing levels of Bcl-2, Bcl-xL, and PARP in P815, C2, and BR cells treated with various concentrations of AR-42 or 1 μΜ 17-AAG for 24 hours;

[0010] FIG. 2 is a western blot showing acetylated H3, H4, and -tubulin in P815, C2, and BR cell lines and canine BMCMCs that were treated with AR-42 and 17-AAG for 24 hours;

[0011 ] FIG. 3 (a) is a western blot showing phosphorylated Kit and total Kit in P815 , C2, BR and canine BMCMCs that were treated with AR-42 and 17-AAG for 24 hours;

[0012] FIG. 3(b) shows the percent of Kit expression using flow cytometry in P815, C2, and BR cells that were treated with 1 μΜ AR-42; [0013] FIG. 3(c) shows the results of quantitative rtPCR for c-Kit was performed on P815, C2, and BR cells treated with AR-42;

[0014] FIG. 4(a) is a western blot for Kit and HSP90 in P815, C2, and BR cells that were treated with 1 or 3 μΜ AR-42 or 1 μΜ of 17-AAG for 8 hours and an untreated control;

[0015] FIG. 4(b) is a western blot for induced HSP70, HSP90 and β-actin in P815, C2 and BR cells that were treated with increasing concentrations of AR-42 and 1 μΜ of 17-AAG for 24 hours;

[0016] FIG. 4(c) is a western blot for acetyl-lysine, total HSP90, and acetyl-tubulin in P815, C2, and BR cells that were treated with 1 or 3 (P815) μΜ AR-42 or 17-AAG for 24 hours;

[0017] FIG. 4(d) is a western blot showing acetyl-lysine in P815, C2, and BR cells that were treated with AR-42 or ΙμΜ 17-AAG for 24 hours;

[0018] FIG. 5 is a western blot for phosphorylated and total levels of Akt, STAT3, and STAT5 in P815, C2, BR and canine BMCMCs that were treated with AR-42 or 17-AAG for 24 hours;

[0019] FIG. 6 shows the percent of C2 cells invading and migrating into the lower chambers after pre-treatment with 1 μΜ AR-42 or 17-AAG;

[0020] FIG. 7(a) shows the percentage of cell viability of malignant mast cells cultured with DMSO, 1 μΜ AR-42 or 17-AAG for 48 hours compared to a control;

[0021] FIG. 7(b) shows the activation of caspases-3/7 for malignant mast cells treated with or without Ι Μ AR-42 or 17-AAG for 48 hours;

[0022] FIG. 7(c) shows surface Kit expression evaluated by flow cytometry of malignant canine mast cells treated with or without 1 μΜ AR-42 and 17-AAG for 24 hours; and

[0023] FIG. 7(d) is a western blot showing the levels of acetylated H3, H4, and p21 in canine malignant mast cells treated with or without 1 μΜ AR-42 or 17-AAG for 24 hours. DETAILED DESCRIPTION

[0024] It is to be understood that the following detailed description are exemplary and explanatory only and are not restrictive of the claims. The accompanying drawings, which are incorporated in and constitute a part of this specification are illustrative and together with the description, serve to explain aspects of the claims.

[0025] In various aspects described herein, the growth of malignant mast cells in an animal, such as humans, is inhibited or prevented by administering a therapeutically effective amount of the various HDAC inhibitors described herein. "Administering" or "administer" means to prescribe or provide a medication comprising an HDAC inhibitor alone or in combination with other active ingredients and inactive ingredients by any suitable route of administration (e.g., orally, parenterally (IV, IM, depot-IM, SQ, and depot-SQ), sublingually, intranasally (inhalation), intrathecally, topically, or rectally). Treatment with the HDAC inhibitors described herein affect mast cell viability, cycling, and signaling. Furthermore, treatment with the HDAC inhibitors described herein induces growth inhibition, cell cycle arrest, apoptosis, activation of caspase 3/7, and promote hyperacetylation of H3, H4 and alpha-tubulin, and upregulation of p21 in malignant mast cells. Downregulation of Kit occurs following treatment via inhibition of Kit transcription. Disassociation between Kit and HSP90 and upregulation of HSP70 occurs after treatment with the HDAC inhibitors described herein, suggesting loss of HSP90 chaperone function. The HDAC inhibitors described herein also downregulate the expression of pAkt, total Akt, pSTAT3/5, and total STAT3/5. The HDAC inhibitors described herein exhibit in vitro and ex vivo biologic activity against malignant mast cells, representing a therapeutic approach for treating malignant mast cell disease.

[0026] The HDAC inhibitors used in the various aspects describe herein include, but are not limited to, the molecules described in U.S. Application No. 10/597,022, which is hereby incorporated by reference in its entirety, and are based on, for example, fatty acids coupled with Zn²⁺-chelating motifs through aromatic Ω-amino acid linkers. In various aspects, the HDAC inhibitors may have the formula:

wherein X is chosen from H and CH₃; Y is (CH₂)n wherein n is 0-2; Z is chosen from (CH₂)_m wherein m is 0-3 and (CH)₂; A is a hydrocarbyl group; B is o-aminophenyl or hydroxyl group; and Q is a halogen, hydrogen, or methyl. One HDAC inhibitor of particular interest is named N-hydroxy- 4-(3-methyl-2-phenyl-butyrylamino)-benzamide, and is also known as AR-42. The formula of AR- 42 is as follows:

[0027] Epigenetic changes are common in many cancers. Unlike genetic changes, epigenetic changes are reversible and evidence suggests that strategies to alter these epigenetic changes have therapeutic potential. Inhibition of histone deacetylation is one approach to modify the expression of various genes that control cell proliferation and survival. Hyperacetylation of histones occurs in both malignant mast cell lines, as well as fresh malignant mast cells following AR-42

treatment. AR-42 induces downregulation of Kit expression in all cell lines through both transcriptional downregulation and loss of chaperone (HSP90) activity.

[0028] Hyperacetylation of histones H3 and H4 is an important biomarker for HDAC inhibition and is necessary for restoration of gene expression. While hyperacetylation of H3 and H4 occurred in all cell lines and tumor samples tested after AR-42 treatment, upregulation of the cyclin-dependent kinase inhibitor p21 was observed only in the C2 line, canine BMCMCs and MCT patient #17 presumably resulting in cell cycle arrest at the Gl phase. [0029] In one aspect, methods of inhibiting or preventing the growth of mast cell tumor cells comprising administering an effective amount of AR-42 to mast cell tumors wherein the growth of malignant mast cells is reduced by about fifteen, twenty, fifty, eighty, or ninety percent or a range there between are provided.

[0030] In another aspect, a method of inducing apoptosis in malignant mast cells is provided comprising administering an effective amount of AR-42 to malignant mast cells wherein apoptosis is induced by about thirty, thirty-five, forty-five, fifty, fifty-five, eighty, or eighty-five percent or a range there between.

[0031] In another aspect, a method of inhibiting the metastases in malignant mast cells is provided comprising administering an effective amount of AR-42 to malignant mast cells wherein metastases is inhibited by about 80 percent, 85 percent, 90 percent, or more.

[0032] In another aspect, a method of decreasing the activity of the Kit oncoprotein comprising administering an effective amount of AR-42 to Kit activated tumors is provided where Kit activity is reduced by at least about twenty-five, fifty, eighty-five, or ninety percent or a range there between.

[0033] In another aspect, both wild-type Kit and various forms of constitutively activated Kit found in canine and mouse malignant mast cells were down-regulated following AR-42 treatment. In contrast, Kit small molecule inhibitors often exhibit variable potencies against specific Kit mutants. For example, imatinib mesylate (Gleevec®) exhibits minimal activity against human malignant mast cells possessing the D816V Kit mutation. Similarly, the concentration of toceranib phosphate (Palladia™) necessary to inhibit the D814V Kit mutant in P815 is 2.5-5 times higher than that sufficient to inhibit Kit possessing a juxtamembrane ITD. In contrast, HDAC inhibitors, particularly AR-42, may exhibit broader efficacy against tumor cells expressing diverse forms of mutant Kit, thereby potentially circumventing issues with drug resistance recognized with typical Kit small molecular inhibitors.

[0034] Downregulation of Kit was found to be at least partly due to inhibition of cKit gene transcription. Furthermore, a reduction in HSP90 chaperone activity may contribute to and possibly enhance the observed loss of Kit expression. Upregulation of inducible HSP70, a biomarker of HSP90 inhibition, occurred following AR-42 treatment, indirectly supporting the notion that HSP90 activity was repressed in the malignant mast cells. Previous studies have demonstrated that HSP90 is a potential target of HDAC inhibitors, resulting in acetylation of HSP90, loss of chaperone function and subsequent degradation of client proteins.

[0035] In another aspect, methods of decreasing the activity of Bcr-Abl²⁵, Her2/Neu, B-Raf and Akt by treating tumors activated by these oncogenes with AR-42 are provided. AR-42 treatment disrupted the protein-protein interaction between HSP90 and Kit, inducing its degradation. It is believed that inhibition of HDAC6, an HSP90 deacetylase, leads to the hyperacetylation of HSP90, with subsequent dissociation from co-chaperones and loss of chaperone activity. In the present study, however, increased acetylation of HSP90, HSP70 or its co-chaperones, HOP and p23, was not detected in AR-42 -treated P815, BR and C2 cells, although tubulin acetylation was evident. AR-42 modulated the activation status and/or expression of several cellular proteins including Akt, STAT3 and STAT5. With respect to the loss of pAkt, this is likely secondary to loss of total Akt, possibly due to inhibition of HSP90 chaperone activity..

[0036] In particular, Akt inhibition was consistent with previous studies suggesting HDAC inhibitors can modulate this protein through several mechanisms. For example, downregulation of Akt can be achieved through loss of HSP90 chaperone activity as it is known client protein. Additionally, AR-42 can induce dephosphorylation of Akt through non-epigenetic mechanisms. Specifically, AR-42 binds to HDAC6, causing its dissociation from protein phosphatase 1 , which then is free to dephosphorylate Akt without changes to total Akt in prostate cancer cells. In another aspect, AR-42 can be administered in combination with other HDAC inhibitors and with Akt inhibitors including, but not limited to, the Akt inhibitors described in U.S. Patent

Application No. 10/957,925, the disclosure of which is incorporated by reference in its entirety. In another aspect, AR-42 can be administered in combination with the Akt inhibitor AR-12, having the formula shown below:

[0037] Downregulation of both p-STAT5 and STAT5 was observed following AR-42 treatment of canine malignant mast cell lines. Previous studies with different HDAC inhibitors demonstrated a decrease in p-STAT5 in human leukemia cells following treatment, but total STAT5 remained unchanged. As described below, both AR-42 and 17-AAG reduced p- STAT5/STAT5 in the malignant mast cells suggesting that this transcription factor may be an HSP90 client protein in mast cells. HDAC inhibitors are known to modulate Akt via several mechanisms. For example, downregulation of total Akt by HDAC inhibitors can be achieved through loss of HSP90 chaperone activity as it is a known client protein. Both pAkt/Akt were reduced in canine mast cell lines. Additionally, AR-42 can also bind HDAC6 causing its dissociation from protein phosphatase 1 , which is then free to dephosphorylate Akt.

[0038] As described below, AR-42 inhibited the migration of C2 cells through Matrigel. While Kit is likely important in tumor cell migration, hyperacetylation of a-tubulin has been reported to perturb microtubule dynamics resulting in the inhibition of migration. Thus, both downregulation of Kit and hyperacetylation of α-tubulin may play a role in the effects of AR-42 on C2 cell motility.

[0039] AR-42 downregulates wild-type and mutant Kit in normal and malignant mast cells, resulting in cell death. Both alteration of cKit gene expression and loss of HSP90 chaperone activity contribute to the observed loss of Kit expression. Without being bound by theory, the effects of AR-42 on multiple cell signaling proteins such as Akt and STAT3 likely enhances the loss of Kit-driven survival signals present in the normal and malignant mast cells. As such, HDAC inhibition is a therapeutic approach for the treatment of malignant mast cell disease. [0040] The HDAC inhibitors described herein may be administered orally, parenterally (IV, IM, depot-IM, SQ, and depot-SQ), sublingually, intranasally (inhalation), intrathecally, topically, or rectally. Dosage forms known to those of skill in the art are suitable for delivery of the HDAC inhibitors described herein.

[0041] Therapeutically effective amounts of the HDAC inhibitors described herein can be formulated into suitable pharmaceutical preparations such as tablets, capsules, or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. The HDAC inhibitors described herein can be formulated into pharmaceutical compositions using techniques and procedures well known in the art.

[0042] In one aspect, about 0.1 to 1000 mg of an HDAC inhibitor or mixture of HDAC inhibitors, or a physiologically acceptable salt or ester is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The HDAC inhibitors can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg, or about 10 to about 100 mg of the active ingredient. The term "unit dosage from" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0043] To prepare compositions, one or more HDAC inhibitors employed in the methods discussed herein are mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the HDAC inhibitors, the resulting mixture can be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for inhibiting or preventing the growth of malignant mast cells in an animal, and may be empirically determined. The HDAC inhibitors can be formulated for single dosage administration.

[0044] Pharmaceutical carriers or vehicles suitable for administration of the HDAC inhibitors provided herein include any such carriers suitable for the particular mode of administration. In addition, the HDAC inhibitors can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The compounds can be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients including, but not limited to, AKT inhibitors such as AR-12.

[0045] Where the HDAC inhibitors exhibit insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using co-solvents such as dimethylsulfoxide (DMSO), using surfactants such as TWEEN, and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs, may also be used in formulating effective pharmaceutical compositions.

[0046] The HDAC inhibitors employed in the methods described herein may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. The HDAC inhibitors can be included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration can be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder.

[0047] The HDAC inhibitors can be enclosed in multiple or single dose containers. The enclosed HDAC inhibitors can be provided in kits, for example, including component parts that can be assembled for use. For example, an HDAC inhibitor in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. A kit can include an HDAC inhibitor and a second therapeutic agent for co-administration. The HDAC inhibitor and second therapeutic agent can be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the HDAC inhibitors employed. The containers can be adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.

[0048] The concentration of active HDAC inhibitor in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. The HDAC inhibitor can be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0049] If oral administration is desired, the HDAC inhibitors can be provided in a

composition that protects it from the acidic environment of the stomach. For example, the HDAC inhibitors can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The HDAC inhibitors can also be formulated in combination with an antacid or other such ingredient.

[0050] Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the HDAC inhibitors can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition. [0051 ] The tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[0052] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The HDAC inhibitors can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup can contain, in addition to the HDAC inhibitors, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[0053] The HDAC inhibitors can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action. The compounds can be used, for example, in combination with an antitumor agent, a hormone, a steroid, or a retinoid. The antitumor agent may be one of numerous chemotherapy agents such as an alkylating agent, an antimetabolite, a hormonal agent, an antibiotic, colchicine, a vinca alkaloid, L-asparaginase, procarbazine, hydroxyurea, mitotane, nitrosoureas or an imidazole carboxamide. Suitable agents include those agents which promote depolarization of tubulin. Examples include colchicine and vinca alkaloids, including vinblastine and vincristine.

[0054] Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

[0055] Where administered intravenously, suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known in the art.

[0056] The HDAC inhibitors may be prepared with carriers that protect the compound against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and

microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.

[0057] The HDAC inhibitors may be administered enterally or parenterally. When administered orally, the HDAC inhibitors can be administered in usual dosage forms for oral administration as is well known to those skilled in the art. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds employed in the methods described herein need to be administered only once or twice daily.

[0058] The oral dosage forms can be administered to the patient 1, 2, 3, 4, or more times daily. Whatever oral dosage form is used, they can be designed so as to protect the HDAC inhibitors from the acidic environment of the stomach. Enteric coated tablets are well known to those skilled in the art. In addition, capsules filled with small spheres each coated to protect from the acidic stomach, are also well known to those skilled in the art. The HDAC inhibitors may also be advantageously delivered in a nanocrystal dispersion formulations.

[0059] The terms "therapeutically effective amount" and "therapeutically effective period of time" are used to denote treatments at dosages and for periods of time effective to reduce neoplastic cell growth, for example, malignant mast cells. When administered systemically, the HDAC inhibitors can be administered at a sufficient dosage to attain a blood level of the HDAC inhibitors of from about 0.1 μΜ to about 100 mM. For localized administration, much lower concentrations than this can be effective, and much higher concentrations may be tolerated. One of skill in the art will appreciate that such therapeutic effect resulting in a lower effective concentration of the histone deacetylase inhibitor may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated. It is also understood that while a patient may be started at one dose, that dose may be varied overtime as the patient's condition changes.

[0060] It should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend on the particular compounds employed, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular patient, and other medication the individual may be taking as is well known to administering physicians who are skilled in this art.

HDAC INHIBITORS

[0061] HDAC inhibitors that can be used as described herein include, but are not limited to, the molecules described in U.S. Application No. 10/597,022, which is hereby incorporated by reference in its entirety, and are based on structural modification of fatty acids, including the short-chain fatty acids, e.g., valproate, butyrate, phenylacetate, and phenylbutyrate. The production of the HDAC inhibitors generally includes coupling fatty acids with Zn²⁺-chelating motifs (including, but not limited to, hydroxamic acid and o-phenylene diamine) through aromatic Ω-amino acid linkers and allows the generation of a large library of compounds via the divergent combination of short-chain fatty acids, Ω-amino acids, and a zinc-chelator, such as hydroxamate. [0062] The fatty acids that can be used herein comprise a hydrocarbyl portion and a carboxylic acid portion. As used herein, the term "hydrocarbyl" is understood to include

"aliphatic," "cycloaliphatic," and "aromatic." The hydrocarbyl groups are understood to include alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, and alkaryl groups. Further, "hydrocarbyl" is understood to include both non-substituted hydrocarbyl groups, and substituted hydrocarbyl groups, with the latter referring to the hydrocarbon portion bearing additional substituents, besides carbon and hydrogen. Additionally, while "carboxylic acid" is used to refer to the compounds, salts of such acids, i.e., carboxylates, are also expressly contemplated. Moreover, carboxylic acids and carboxylates may be used interchangably herein.

[0063] In particular, fatty acids include, but are not limited to, those having chain lengths comparable to an unbranched fatty acid of from about 3 carbons to about 14 carbons in length. Thus, the chains can be, for example, from about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbons in length. The chains can be up to, for example, about 14, 13, 12, 11, 9, 8, 7, 6, 5, or 4 carbons in length. The fatty acids can be straight or branched and can include single, double, and/or triple bonds. Nonlimiting examples of fatty acids include valproate, butyrate, phenylacetate, and phenylbutyrate.

[0064] Zinc²⁺-chelating motifs contemplated herein include, but are not limited to, hydroxamic acids and o-phenylene diamines. Other examples include trifiuoromethyl ketone, a- keto amide, a-keto thiazole, 2-keto 1 -methyl- lH-imidazole, a-keto ΙΗ-tetrazole, a-keto 1H- imidazole, 5-keto 1 -methyl- lH-imidazole, a-keto oxazole, a-keto 4,5-dihydro-oxazole, a-keto benzooxazole, a-keto oxazolo[4,5-b]pyridine, and a-keto pyridine.

[0065] The spacer can be any hydrocarbyl spacer, but preferably comprises an aromatic component. Aromatic linkers are believed to possess the following advantages: 1) they enhance the structural rigidity of the conjugate, and 2) they increase van der Waals contacts with the tubelike hydrophobic region of the pocket to improve binding affinity. Examples of linkers include, but are not limited to, aromatic Ω-amino acids.

[0066] The linkers can exhibit lengths equivalent to that of four to eight-carbon straight chains, e.g., equivalent to 4, 5, 6, 7, or 8-carbon straight chains. Thus, the lengths can be equivalent to 4-7 or 4-6-carbon straight chains. The length may be based on the depth of the hydrophobic region of the binding pocket. Examples of linkers include, but are not limited to, 4- (aminomethyl)benzoic acid, 4-aminobenzoic acid, (4-aminophenyl)acetic acid, 3-(4- aminophenyl)propionic acid, and 3-(4-aminophenyl)-acrylic acid. Among them, 4- (aminomethyl)benzoic acid has been used as the linker for MS-27-275.

[0067] In one aspect, the HDAC inhibitors used to treat malignant mast cells have the formula:

wherein:

[0068] X is chosen from H and CH₃;

[0069] Y is (CI¾)n wherein n is 0-2;

[0070] Z is chosen from (CH₂)_m wherein m is 0-3 and (CH)₂;

[0071] A is a hydrocarbyl group;

[0072] B is o-aminophenyl or hydroxyl group; and

[0073] Q is a halogen, hydrogen, or methyl.

[0074] In this aspect, A can comprise an aliphatic group, and the aliphatic group can be branched. A can also comprise an aromatic group, which may be substituted or unsubstituted. In the above formula, B can be o-aminophenyl or hydroxyl. In some embodiments Y can be (CH2)_n wherein n is 0, A comprises an aromatic group, B is hydroxy, and Q is hydrogen.

[0075] In another aspect, m is 0 and X is H. Compounds having these features include, but are not limited to,

which is also known as N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide or AR-42,

[0076] In some specific embodiments, X is H and A is chosen from:

wherein R comprises a branched or unbranched, substituted or unsubstituted, saturated or unsaturated, aliphatic or aromatic group. [0077] In various embodiments, the HDAC inhibitors may be enriched in the S-stereoisomer as compared to the R-stereoisomer. The HDAC inhibitors may also be part of a pharmaceutical compositions including at least one pharmaceutically acceptable excipient.

[0078] The following compounds are specifically contemplated:

[0079] N-(2-Amino-phenyl)-4-[(2-propyl-pentanoylamino)-methyl]-benzamide;

[0080] N-Hydroxy-4- [(2-propy 1-pentanoy lamino)-methyl] -benzamide ;

[0081] N-(2-Amino-phenyl)-4-(2-propyl-pentanoylamino)-benzamide;

[0082] N-Hydroxy-4-(2-propyl-pentanoylamino)-benzamide;

[0083] 2-Propy 1-pentanoic acid {4- [2-amino-phenylcarbamoy l)-methy 1] -phenyl } -amide ;

[0084] 2-Propyl-pentanoic acid (4-hydroxycarbamoyl-methyl-phenyl)-amide;

[0085] 2-Propyl-pentanoic acid {4-[2-amino-phenylcarbamoyl)-ethyl]-phenyl}-amide;

[0086] 2-Propyl-pentanoic acid [4-(2-hydroxycarbamoyl-ethyl)-phenyl]-amide;

[0087] 2-Propyl-pentanoic acid {4-2-(2-amino-phenylcarbamoyl)-vinyl]-phenyl}-amide;

[0088] 2-Propyl-pentanoic acid [4-(2-hydroxy carbamoyl- viny l)-pheny 1] -amide ;

[0089] N-(2-Amino-phenyl)-4-(butyrylamino-methyl)-benzamide;

[0090] N-(2-Amino-phenyl)-4-(phenylacetylamino-methyl)-benzamide;

[0091] N-(2-Amino-phenyl)-4-[(4-phenyl-butyrylamino-methyl]-benzamide;

[0092] 4-(Butyrylamino-methyl)-N-hydroxy-benzamide;

[0093] N-hydroxy-4-(phenylacetylamino-methyl)-benzamide;

[0094] N-hydroxy-4- [(4-phenyl-butyry lamino)-methy 1] -benzamide ;

[0095] 4-Butyry lamino-N-hydroxy-benzamide ;

[0096] N-hydroxy-4-phenylacetylamino-benzamide;

[0097] N-hydroxy-4-(4-phenylbutyrylamino)-benzamide; [0098] N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-butyramide;

[0099] N-hydroxy-3 -(4-pheny lacetylamino-phenyl)-propionamide;

[00100] N- [4-(2-Hydroxycarbamoyl-ethy l)-pheny 1] -4-pheny 1-butyramide ;

[00101] N-(2-Amino-phenyl)-4-[(2-phenyl-butyrylamino-n ethyl]-benzamide;

[00102] N-(2-Amino-phenyl)-4-[(3-phenyl-butyrylamino-methyl]-benzamide;

[00103] N-hydroxy-4-(2-phenylbutyrylamino)-benzamide ;

[00104] N-hydroxy-4-(3-phenylbutyrylamino)-benzamide;

[00105] N- [4-(2-Hydroxycarbamoyl-ethy l)-pheny 1] -2-phenyl-butyramide ;

[00106] N- [4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-3 -phenyl-butyramide;

[00107] N-hydroxy-4- [(2-phenyl-butyrylamino)-methy 1] -benzamide;

[00108] N-hydroxy-4- [(3 -phenyl-butyrylamino)-methyl] -benzamide;

[00109] 4-Benzoylamino-N-hydroxy-benzamide;

[00110] 4-(4-methyl)-Benzoylamino-N-hydroxy-benzamide;

[00111] 4-(4-chloro)-Benzoylamino-N-hydroxy-benzamide;

[00112] 4-(4-bromo)-Benzoylamino-N-hydroxy-benzamide;

[00113] 4-(4-tert-butyl)-Benzoylamino-N-hydroxy-benzamide;

[00114] 4-(4-phenyl)-Benzoylamino-N-hydroxy-benzamide;

[00115] 4-(4-methoxyl)-Benzoylamino-N-hydroxy-benzamide;

[00116] 4-(4-trifluoromethyl)-Benzoylamino-N-hydroxy-benzamide;

[00117] 4-(4-nitro)-Benzoylamino-N-hydroxy-benzamide;

[00118] Pyridine-2-carboxylic acid (4-hydroxycarbamoyl-phenyl)-amide;

[00119] N-hydroxy-4-(2-methyl-2-phenyl-propionylamino)-benzamide;

[00120] N-hydroxy-4-(3 -methyl-2-phenyl-butyrylamino)-benzamide; [00121] N-hydroxy-4-(3-phenyl-propionylamino)-benzamide;

[00122] 4-(2,2-Dimethyl-4-phenyl-butyrylamino)-N-hydroxy-benzamide;

[00123] N-hydroxy-4-[methyl-(4-phenyl-bu1yryl)-an ino]-benzamide;

[00124] N-hy droxy-4-(2-phenyl-propionylamino)-benzamide ;

[00125] N-hydroxy-4-(2-methoxy-2-phenyl-acetylamino)-benzamide;

[00126] 4-Diphenylacetylamino-N-hydroxy-benzamide;

[00127] N-hydroxy-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzamide;

[00128] N-(2-Amino-phenyl)-4-phenylacetylamino-benzamide;

[00129] N-(2- Amino-phenyl)-4-(5-phenyl-pentanoylamino)-benzamide;

[00130] N-(2-Amino-phenyl)-4-(2-phenyl-butyrylamino)-benzamide;

[00131 ] N-(2-Amino-phenyl)-4-(2,2-dimethyl-4-phenyl-butyrylamino)-benzamide- ;

[00132] N-(2-Amino-phenyl)-4-(3-phenyl-propionylamino)-benzamide;

[00133] N-(2-Amino-phenyl)-4-(4-phenyl-butyrylamino)-benzamide;

[00134] N-(2-Amino-phenyl)-4-(3-phenyl-butyrylamino)-benzamide;

[00135] N-(2- Amino-phenyl)-4-(3 -methyl-2-phenyl-butyrylamino)-benzamide;

[00136] N-(2- Amino-phenyl)-4-(2-methyl-2-phenyl-propionylamino)-benzaniide;

[00137] N-(2-Amino-phenyl)-4-[2-(4-isobutyl-phenyl)-propionylamino]-benzami- de;

[00138] N-hydroxy-4- [2-(S)-phenylbutyrylamino] -benzamide;

[00139] N-hydroxy-4- [2-(R)-phenylbutyry lamino] -benzamide ;

[00140] N-[4-(2-Hydroxycarbamoyl-ethyl)-phenyl]-2-(S)-phenyl-butyramide;

[00141 ] N- [4-(2-Hydroxycarbamoyl-ethyl)-phenyl] -2-(R)-phenyl-butyramide;

[00142] N-hydroxy-4-(3 -(S)-phenylbutyrylamino)-benzamide;

[00143] N-hydroxy-4-(3-(R)-phenylbutyrylamino)-benzamide; [00144] N-hydroxy-4-[3-(S)-phenylbutyrylamino]-benzamide; and

[00145] N-hydroxy-4-[3-(R)-phenylbutyrylamino]-benzamide.

[00146] The HDAC inhibitors described herein may be in the form of esters and salts. The HDAC inhibitors described herein can also be racemates, or racemic mixtures. The term

"racemic" as used herein means a mixture of the (R)- and (S)-enantiomers, or stereoisomers, of the HDAC inhibitors, in which neither enantiomer, or stereoisomer, is substantially purified from the other.

EXAMPLES

[00147] AR-42 induces apoptosis in both mouse (P815) and canine (C2 and BR) malignant mast cell lines. Cells from P815, C2, and BR lines were treated with AR-42 for 24 hours, after which the cell proliferation rate was assessed using a BrdU incorporation assay. Three independent experiments were performed, and one representative result is presented. As shown in FIG. 1(a), AR-42 inhibited cell proliferation in a dose dependent manner in all three lines. The IC₅₀ concentrations for P815, C2 and BR cells were 0.65, 0.30, and 0.23 μΜ, respectively. To determine if AR-42 treatment induced cell death, effects of drug treatment on cell cycle and apoptosis were analyzed. P815, C2 and BR cells were treated with 0.5 μΜ AR-42 for 24 hours. Cells were then evaluated for effects on cell cycle using propidium iodide staining and flow cytometry. Three independent experiments were performed, and one representative result is presented. As shown in FIG. 1(b), AR-42 induced cell cycle arrest at Gl in the P815 cells and at G1/G2 in the C2 cells at a concentration of 0.5μΜ. BR cells failed to show significant cell cycle arrest, but a large number of dead cells (sub Gl) were noted. P815, C2, and BR cells (10 x 10⁶) were then treated with increasing concentrations of AR-42 and 1 μΜ 17-AAG for 24 hours.

Apoptosis was assessed by AnnexinV/PI staining and flow cytometry (*P<0.05). Three independent experiments were performed and data were calculated from the three experiments. As shown in FIG. 1(c), AR-42 caused a dose-dependent induction of apoptosis as determined by enhanced labeling with annexin/PI. P815, C2, and BR cells were incubated with various concentrations of AR-42 and 17-AAG for 24 hours and caspase 3/7 activation was assessed (*P<0.05). Experiments were performed in triplicate and repeated three times. As shown in FIG. 1(d), there was a significant increase in casepase3/7 activity. P815, C2, and BR cells were treated with various concentrations of AR-42 or 1 μΜ 17-AAG for 24 hours. Effects on the expression of Bcl-2, Bcl-xL, and PARP were determined by Western blot analysis, as shown in FIG. 1(e). As shown in FIG. IF, expression levels of Bcl-xL and Bcl-2 were unaffected by AR-42 treatment, although the BR cells express undetectable levels of Bcl-2 at baseline. These findings suggest that, in P815, C2 and BR malignant mast cells, AR-42 does not alter the expression of Bel family member genes and that apoptosis induced by treatment with the drug may be independent of this pathway. 17-AAG, which was used as the positive control induced apoptosis in all cell lines.

[00148] AR-42 induces hyperacetylation of histones H3 and H4 and a-tubulin in malignant mast cells. Biomarkers of HDAC inhibition include hyperacetylation of histones and a-tubulin, and the up-regulation of p21 expression. The effects of AR-42 on these biomarkers were assessed in P815, C2 and BR cell lines and canine BMCMCs. P815, C2, and BR cell lines were treated with the indicated concentrations of AR-42 or 1 μΜ of 17-AAG and canine BMCMCs were treated with 1 μΜ of AR-42 or 17-AAG for 24 hours. As shown in FIG. 2, a dose dependent hyperacetylation of histone H3, histone H4, and α-tubulin was observed in all cell lines as well as the normal canine mast cells. While upregulation of p21 was noted in the C2 line and canine BMBMCs, this did not occur in the P815 and BR cells after exposure to AR-42. However, P815 cells exhibited a higher basal level of p21 expression relative to C2 cells, while the p21 level in BR cells was undetectable regardless of treatment. As expected, the HSP90 inhibitor 17-AAG did not induce hyperacetylation of histones or α-tubulin in treated cells.

[00149] AR-42 downregulates the expression of mutant Kit via downregulation of Kit mRNA transcript levels. Canine BMCMCs express wild-type Kit and are highly SCF-dependent for their survival and proliferation. In contrast, malignant mast cell lines and spontaneous primary malignant mast cell tumors often possess activating mutations in Kit including catalytic domain point mutations (D814V/D816V mouse/human cell lines and human systemic mastocytosis) and juxtamembrane domain ITDs and point mutations (dog mast cell tumors and dog mast cell lines). These gain-of-function mutations in Kit are important mediators of proliferation, migration and survival and represent a relevant target for therapeutic intervention. P815, C2, and BR cells and canine BMCMCs were treated with either AR-42 and 17-AAG (ΙμΜ AR-42 and 17-AAG for BMCMCs) at the indicated concentrations for 24 hours. Western blotting analysis was performed for pKit/total Kit and demonstrated reduced levels of phosphorylated and total Kit in all cell lines and canine BMCMCs (wild type) after 24 hours of treatment with AR-42, as shown in FIG. 3(a). The upper band represented mature form of Kit and the lower band represented the immature form of Kit. 17-AAG, used as the control for these experiments, was previously shown to downregulated various forms of constitutively activated Kit in malignant mast cell lines. The canine BMCMCs were maintained in the presence of 50 ng/ml rcSCF resulting in the observed phosphorylation of wild-type Kit. Next, P815, C2, and BR cells (1.0 x 10⁶) were treated with 1 μΜ AR-42. Cells were collected after 6 hours after drug treatment, and evaluated for Kit expression by flow cytometry. PE conjugated rat-IgG Ab was used as isotype control. Three independent experiments were performed, and one representative result is shown. As shown in FIG. 3(b), in P815, C2 and BR cells, Kit cell surface expression was downregulated following only 6 hours of AR-42 treatment. P815, C2, and BR cells were treated with AR-42 at the indicated concentrations and were collected at 4 and 8 hours after treatment and quantitative rtPCR for c-Kit was performed to determine if modulation of Kit protein expression was secondary to loss of cKit mRNA. Experiments were performed in triplicate and repeated three times. The difference between treatment groups and DMSO control group was analyzed using the student t-test and a *p-val e less than 0.05 was considered significant. As shown in FIG. 3(c), it was determined that AR-42 downregulated cKit mRNA transcription at both time points, indicating that alteration of gene transcription is at least partially responsible for the observed effects of AR-42 on Kit protein levels.

[00150] AR-42 induces disassociation between Kit and HSP90 without evidence of HSP90 hyperacetylation. To examine the effects of AR-42 on the interaction between HSP90 and Kit, P815, C2, and BR cells were treated with 1 or 3 (P815) μΜ AR-42 or ΙμΜ of 17-AAG for 8 hours. HSP90 was immunoprecipitated from the cell lysates and the levels of Kit and HSP90 in the immunoprecipitates were determined by Western blot analysis.. Western blotting for Kit and HSP90 was also performed from total cell lysates (50 μg) prior to immunoprecipitation as a control. As shown in FIG. 4(a), both AR-42 and 17-AAG treatments reduced the amount of Kit associated with HSP90 in all cell lines. Next, P815, C2, and BR cells were treated with increasing concentrations of AR-42 or 1 μΜ 17-AAG for 24 hours. Equal amounts of cell lysates were analyzed by Western blotting to detect induced HSP70, HSP90 and β-actin. As shown in FIG. 4(b), HSP70 protein levels were up regulated after AR-42 treatment, which is indicative of loss of HSP90 activity, while HSP90 expression remained relatively unchanged. However, the magnitude of the HSP70 upregulation was substantially less than that induced by 17-AAG treatment.

[00151] To explore acetylation as a potential mechanism for loss of HSP90 chaperone activity, P815, C2 and BR cells were treated with DMSO (control), 3 μΜ (P815 cells) or 1 μΜ (C2 and BR cells) AR-42 or ΙμΜ 17-AAG for 24 hours, then immunoprecipitated HSP90 from protein lysates (7% SDS-PAGE of 200 μg total protein). Western blotting was performed for acetyl- lysine, and the blots were stripped and re-probed for HSP90, and then for acetyl-tubulin. No evidence of acetyl-lysine was detected, despite demonstrating the presence of HSP90 on the re- probed blots. P815, C2, and BR cells were treated with DMSO, AR-42 or 17-AAG for 24 hours, protein lysates were generated and 7% SDS-PAGE was used to obtain good separation of proteins in the 90 kD size range. Western blotting was performed for acetyl-lysine and the blots were then stripped and re-probed for HSP90. As shown in FIG. 4(c), no acetyl-lysine was evident at 90 kD despite the presence of HSP90 at this location upon re-probe. In contrast, acetylated ot-tubulin was shown to be present in treated cells when the blots were again stripped, then re-probed for acetylated tubulin. Mast cell lines were treated with 3 μΜ (P815 cells) or 1 μΜ (C2 and BR cells) AR-42 or ΙμΜ 17-AAG for 24 hours. Protein lysates were generated and following 10% SDS- PAGE of 200 μg total protein, Western blotting was performed for acetyl-lysine. Multiple acetylated proteins are evident on the Western blot, although none corresponds directly to HSP90. All experiments in this figure were performed three times. As shown in FIG. 4(d), multiple cellular proteins were acetylated following AR-42, but not 17-AAG, treatment. These data indicate that AR-42 is capable of inducing the acetylation of non-histone targets, although HSP90 does not appear to be one of these targets in either the mouse or canine malignant mast cell lines. The HSP90 co-chaperones HSP70, HOP and P23 were not acetylated following AR-42 treatment, which indicates that direct acetylation of the super-chaperone complex is likely not responsible for the observed loss of HSP90 function.

[00152] AR-42 modulates Akt and STAT3/5 pathways in malignant mast cells and alters STAT3 DNA binding. HDAC inhibitors are known to modulate the activity of multiple molecular targets including Akt, and transcription factors, such as STAT3, which are involved in the regulation of mast cell viability and proliferation. P815, C2, and BR cell lines and canine BMCMCs (1 μΜ of AR-42 and 17-AAG) were treated with AR-42 (0.5 - 3 μΜ) or 17-AAG (1 μΜ) for 24 hours. Effects on the expression of phosphorylated and total levels of Akt, STAT3 and STAT5 were determined by Western blotting. Three independent experiments were performed. As shown in FIG. 5, AR-42 reduced levels of pAkt/total Akt and pSTAT3 in C2 and BR cell lines as well as canine BMCMCs, while it also downregulated both pSTAT5/total STAT5 in C2 and BR cells; total STAT3 levels remained unchanged in these cell lines. AR-42 failed to modulate Akt and STAT3 in the P815 cells. As expected, the HSP90 inhibitor 17-AAG induced

degradation of its client proteins Akt, STAT3 and STAT5.

[00153] To assess the effects of AR-42 treatment on malignant mast cell invasion, C2 cells were pre-treated with 1 μΜ AR-42 or 17-AAG for 8 hours and then transferred onto cell culture inserts with Matrigel®. After 20 hours of incubation, cells that had invaded and migrated into the lower chambers were collected and measured by Cyquant® fluorescence. To adjust for treatment- associated cell death, C2 cells were treated in parallel under the same conditions, but without transfer onto cell culture inserts. Cell numbers were determined by CyQuant® assay. These experiments were performed in triplicate and repeated three times. As shown in FIG. 6, cell invasion was significantly suppressed by AR-42 and 17-AAG treatment compared to the DMSO- treated controls.

[00154] AR-42 induces Kit downregulation and caspase-3/7 activation in malignant canine mast cells cultured ex vivo. Canine mast cells share many functional similarities with their human counterparts and serve as a good biological model for human mast cell associated diseases. Although different Kit mutations have been identified in human and canine malignant mast cells, therapies targeting Kit have been proposed, validated and intensively studied in both species. The prevalence of spontaneous canine malignant mast cell disease is high compared to human malignant mast cell disease, and as such, it is relatively easy to obtain fresh tumor specimens for biological analysis. The activity and target modulation of AR-42 was therefore validated using primary canine malignant mast cells derived directly from affected dogs. Malignant mast cells were purified from fine needle aspiration samples of tumors from 17 dogs diagnosed with this disease. The purity of the cells was determinate by cytology and RBCs were removed by hypotonic shock. Cells were treated with DMSO, 1 μΜ AR-42, 17-AAG or DMSO for 24 or 48 hours, and then evaluated for cell viability, caspase 3/7 activity, acetylated histones H3 and H4, and changes in expression of p21, Kit and HSP70 using the WST-1 assay. Data from all 17 tumor samples was pooled and the percentage of cell viability following AR-42 or 17-AAG treatment was calculated using the DMSO group as the 100% control (*P < 0.05). As shown in FIG. 7(a), AR-42 and 17-AAG treatment significantly reduced the viability of fresh malignant mast cells cultured ex vivo after 48 hours. Next, fresh malignant mast cells from four different patients were treated with or without 1 μΜ AR-42 or 17-AAG in triplicate for 48 hours and activation of caspases-3/7 was determined. The difference was analyzed between treatment groups (AR-42 and 17-AAG) and the DMSO control group for each individual tumor sample (*P < 0.05). As shown in FIG. 7(b), the loss of cell viability was associated with significant up-regulation of caspase 3/7 activity suggesting involvement of caspase-dependent cell death. Consistent with findings in the cell lines, AR-42 treatment induced greater caspase 3/7 activity than 17-AAG treatment in the fresh malignant mast cells when compared to cells treated with DMSO alone. Fresh malignant canine mast cells were washed and treated with or without 1 μΜ AR-42 and 17-AAG for 24 hours, and cells were collected and evaluated for surface Kit expression by flow cytometry. As shown in FIG. 7(c), both AR-42 and 17-AAG down-regulated cell surface Kit expression 24 hours post treatment. A total of 3 experiments from 3 different patients was performed once with similar results. Fresh canine malignant mast cells and normal canine BMCMCs from four different patients were treated with or without 1 μΜ AR-42 or 17-AAG for 24 hours. The cells were collected, lysed and following SDS-PAGE of 50 μg total protein, western blotting for acetylated H3, H4, a-tubulin and β-actin was performed. As shown in FIG. 7(d), hyperacetylation of histones H3 and H4 occurred after AR-42 treatment in the fresh malignant mast cells, as was observed previously in normal canine BMCMCs, but p21 expression was largely unchanged in most of the ex vivo samples. As with the mast cell lines, 17-AAG treatment did not affect

acetylation of histones in the fresh malignant mast cells.

Reagents, cell lines and fresh tumor samples

[00155] The HDAC inhibitor, AR-42, ((S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl- butyrylamino)benzamide), was synthesized with purities exceeding 99% as determined by nuclear magnetic resonance spectroscopy (300 MHz). 17-AAG was provided by Synta Pharmaceuticals (Lexington, MA). The following antibodies were obtained from Cell Signaling Technologies

(Danvers, MA): p-Kit (Tyr719), Kit, p-Akt (Ser473 and Tyr308), p-STAT3 (Tyr705), p-STAT5 (Tyr694), STAT3, STAT5, acetylated lysine, HSP70, and HSP90. A monoclonal antibody against acetylated tubulin (Clone6-l lB-1) and Stemline® serum-free medium were obtained from Sigma (St. Louis, MO). Antibodies against Akt, Bcl-2, and PARP were obtained from BD Biosciences

(Franklin Lakes, NJ). BC1-XL/S, HSP90a/p, and β-actin antibodies were from Santa Cruz

Biotechnology, (San Diego, CA). Kit antibody was obtained from Calbiochem, (San Diego, CA). Monoclonal antibodies against HOP (HSP organizer protein) and p23 were purchased from Stressgen (Ann Arbor, MI) and Abeam (Cambridge, MA), respectively. Monoclonal anti-constitutive HSP70 (HSP70c) antibody was provided by Dr. Michael Oglesbee (Ohio State Universtiy, OH). Antibodies against acetylated histones H3 (Lys 9 and 14) and H4 (Lys 5, 8, 12, and 16) were obtained from Millipore (Billerica, MA).

[00156] Mouse P815 (activating D814V Kit mutation - homologous to the human KIT D816V mutation found in the HMC-1 cell line) and canine C2 (activating internal tandem duplication, ITD, mutation in the JM domain of Kit) and BR (activating point mutation L575P in the JM domain of Kit) cells were provided by Dr. Stephen Galli (Stanford University, CA) and Dr. Warren Gold (University of California, San Francisco, CA) and were maintained in RPMI1640 with 10% FBS and antibiotics. Canine bone marrow derived cultured mast cells (canine BMCMCs) were generated from two different dogs and maintained in Stemline® medium supplemented with recombinant canine SCF as previously described. Fine needle aspirates were performed on spontaneously occurring canine MCTs in order to obtain small numbers of primary malignant mast cells and processed as previously detailed resulting in 60-95% purity. Aspirates were obtained from 17 different affected dogs presented to the Veterinary Teaching Hospital at The Ohio State University in accordance with a protocol approved by the hospital Clinical Trials Advisory Committee.

Assessment of proliferation and cell viability

[00157] Changes in cell proliferation were assessed using a commercially available BrdU incorporation assay (Roche, Switzerland). Briefly, 15xl0⁴ P815, C2, and BR cells were treated with DMSO, AR-42, or 17-AAG as indicated for 24 hours in 96-well plates. The BrdU reagent was added, cells were incubated for another 2-3 hours then harvested, fixed and digested by nuclease for 30 min at 37°C. Cells were then incubated with conjugate for 1 hour, washed, and the plates were developed by adding 100 μΐ of substrate for 30 minutes. The absorbance was measured using an ELISA plate reader (Spectra Max, Molecular Devices, Carlsbad, CA). To measure changes in the viability of primary canine malignant mast cells following drug treatments, the WST-1 assay

(Clontech, San Francisco, CA) was used. Briefly, 2-4 x 10⁵ fresh malignant canine mast cells were treated with 0.1% DMSO, AR-42 or 17-AAG for 48 hours in 96-well plates. The WST-1 working solution was then added, plates were incubated for 4.5 hours and absorbances were measured by ELISA plate reader.

Cell cycle analysis and evaluation of apoptosis

[00158] The effects of drug treatment on cell cycle and apoptosis of canine and mouse mast cell lines were measured by annexin-V/propidium iodide (PI) and PI staining. Briefly, 1.0 x 10⁶ P815, C2 and BR cells were treated with 0.1% DMSO, AR-42 or 17-AAG for 24 hours at 37°C. Cells were collected, washed and stained with annexin-V-FITC and PI for 15 min before evaluation by flow cytometry. Alternatively, cells were treated as above, collected, washed three times in 0.1% glucose/PBS and then fixed with cold 70% EtOH at 4°C overnight. After three 0.1% glucose/PBS washes, 200 μΐ of PI working solution (50μg/ml in PBS) was added prior to analysis by flow cytometry.

[00159] Induction of caspase 3/7 activity was measured using the SensoLyte™ homogeneous

AMC caspase 3/7 assay kit (Anaspec, San Jose, CA). Briefly, 5.0 x 10⁴ C2 and BR cells, 5.0 x 10³

P815 cells and 2-4 x 10⁵ fresh malignant mast cells were treated with DMSO, AR-42, or 17-AAG for 24 hours in 96-well plates, after which the substrate (Ac-DEVD-AMC) was added to each well and plates were incubated for 40 minutes at room temperature. Wells containing equal amounts of medium with 0.1% DMSO and substrate were used as blanks. Fluorescence was measured by ELISA plate reader and the data is presented as relative fluorescence units (RFU).

Western blotting and co-immunoprecipitation

[00160] P815, C2, and BR cells (1.0 x 10⁷ cells) and fresh malignant canine mast cells (1 x 10⁶ cells) were treated with DMSO, AR-42, or 17-AAG for 24 hours. Cells were collected, washed, and lysed in protein lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein lysate were used for SDS-PAGE. Following transfer the membranes were incubated with primary antibodies overnight at 4°C and corresponding HRP-conjugated secondary antibodies for 1 hour at room temperature then developed with Super Signal West Pico-chemiluminescence substrate ( Pierce, Rockford, IL).

[00161] To assess the effects of drug treatment on the association of HSP90 with Kit, 3.0 x 10⁷ P815, C2, and BR cells were treated with DMSO, AR-42, or 17-AAG for 8 hours. Cells were collected and lysates generated as described above. The lysates were then pre-cleared with 50 μΐ of True-blot anti-rabbit agarose® beads (eBioscience, San Diego, CA) for 30 minutes on ice. Rabbit anti-HSP90 antibody (5 μΐ) was added to samples, which were then incubated for another 60 minutes on ice, followed by the addition of 50 μΐ of True-blot anti-rabbit agarose® beads. The samples were incubated at 4°C overnight and then analyzed by western blotting to detect acetyl-lysine, HSP90 or Kit.

Kit cell surface expression

[00162] P815, C2, and BR cells (1.0 x 10⁶) were treated for six hours, and 0.5 x 10⁶ fresh malignant mast cells were treated for 24 hours with ΙμΜ AR-42 or 17-AAG. Cells were harvested, washed and analyzed for Kit cell surface expression by flow cytometry as previously described. For the analysis, only live cells were gated (i.e., cellular debris was gated out).

Quantitative RT-PCR analysis of cKit expression [00163] P815, C2, and BR cells were treated with AR-42 or 17-AAG for 24 hours for 4 and 8 hours and RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was made from 1 μg total RNA using Superscript III (Invitrogen). Real-time quantitative PCR was performed using the Applied Biosystem's 7900HT Sequence Detection System. cKit and 18S were detected using Fast SYBR green PCR master mix (Applied Biosystems) according to the manufacturer's protocol and primer sets detailed in Table 1. All reactions were performed in triplicate and included no-template controls for each gene. Relative expression was calculated using the comparative Ct method.

Experiments were repeated 3 times using samples in triplicate.

Matrigel invasion assay

[00164] To assess the ability of AR-42 to inhibit invasion, a Matrigel® invasion assay using cell culture inserts (8μηι pore size, Falcon, Los Angeles, CA) coated with 100 μΐ of BD Matrigel® (BD Bioscience) was used. Briefly, C2 cells (5 10⁵/ml) were pretreated with DMSO, AR-42, or 17- AAG for 8 hours. Cells were then transferred into the upper chamber of inserts. After 20 hours at 37°C, the cells in the lower chamber were collected and stored at -80°C until cell numbers were determined using the CyQuant assay as previously described (Invitrogen, Carlsbad, CA). To account for cell death following drug treatment, an equivalent number of the C2 cells, treated for the same duration with the same concentration of drugs, served as the baseline comparison for each individual assay.

Statistics

[00165] All experiments with the exception of those involving canine BMCMCs and primary mast cells were performed in triplicate and repeated three times. Experiments using canine BMCMCs were performed in triplicate, but repeated only twice due to limited cell numbers. Experiments using primary malignant mast cells cultured ex vivo were performed in triplicate but only undertaken once given the limited cell numbers and lack of long-term viability in culture. Data was presented as mean ± SD. The difference between two group means was analyzed using the student t-test and a -value less than 0.05 was considered significant.

[00166] Other aspects will be apparent to those skilled in the art from consideration of the specification and practice of the claims. The specification and examples are exemplary only.

Claims

We claim:

1. A method of inhibiting or preventing the growth of malignant mast cells in an

comprising: administering a therapeutically effective amount of a compound having the formula:

wherein:

X is chosen from H and CH₃; Y is (CH₂)n wherein n is 0-2;

Z is chosen from (CH₂)_m wherein m is 0-3 and (CH)₂;

A is a hydrocarbyl group;

B is o-aminophenyl or hydroxyl group; and

Q is a halogen, hydrogen, or methyl to an animal in need of treatment wherein the growth of malignant mast cells is inhibited or prevented.

2. The method according to claim 1, wherein the growth of the malignant mast cells is inhibited or prevented by at least about 20 percent.

3. The method according to claim 1, wherein the growth of malignant mast cells is inhibited or prevented by about 20 to about 80 percent.

4. The method according to claim 1 , wherein A is an aromatic, aralkyl, or alkaryl group having from 5 to 14 carbons.

5. The method according to claim 1, wherein B is hydroxyl.

6. The method according to claim 1, wherein the hydrocarbyl group comprises a branched alkyl group.

7. The method according to claim 1, wherein the compound is:

8. The method of claim 1 , wherein the animal is a human.

9. The method of claim 1, wherein the animal is a canine.

10. The method of claim 1, wherein the malignant mast cell expresses Kit.

11. The method of claim 1 , wherein inhibiting or preventing the growth of malignant mast cells further comprises inducing activation of caspase3/7 in the malignant mast cells.

12. The method of claim 1, wherein hyperacetylation of H3, H4 and alpha-tubulin in the malignant mast cells is promoted.

13. The method of claim 1, wherein p21 is upregulated in the malignant mast cells.

14. The method of claim 1, wherein wild-type and mutant Kit is downregulated by at least twenty-five percent or more in the malignant mast cells.

15. The method of claim 1 , wherein disassociation between Kit and HSP90 is promoted in the malignant mast cells.

16. The method of claim 1, wherein HSP70 is upregulated in the malignant mast cells.

17. The method of claim 1, wherein the expression of pAkt, total Akt, pSTAT3/5, total STAT3/5 and MMP9 is downregulated in the malignant mast cells.

18. A method of inhibiting the metastases of malignant mast cells in an animal comprising: administering a therapeutically effective amount of a compound having the formula:

wherein:

X is chosen from H and CH₃; Y is (CH₂)n wherein n is 0-2;

Z is chosen from (CH₂)_m wherein m is 0-3 and (CH)₂;

A is a hydrocarbyl group;

B is o-aminophenyl or hydroxyl group; and

Q is a halogen, hydrogen, or methyl, to an animal in need of treatment wherein the metastases of malignant mast cells is inhibited by at least about 80 percent.

19. A method of inducing apoptosis in malignant mast cells comprising: administering a therapeutically effective amount of a compound having the formula:

wherein:

X is chosen from H and CH₃; Y is (CH₂)n wherein n is 0-2;

Z is chosen from (CH₂)_m wherein m is 0-3 and (CH)₂;

A is a hydrocarbyl group;

B is o-aminophenyl or hydroxyl group; and

Q is a halogen, hydrogen, or methyl, to an animal in need of treatment wherein apoptosis of malignant mast cells is induced. A method of inhibiting the activity of histone deacetylases within a malignant mast cell comprising: administering a therapeutically effective amount of at least one histone deacetylase inhibitor having the formula:

wherein: X is chosen from H and CH₃;

Y is (CH₂)n wherein n is 0-2;

Z is chosen from (CH₂)_m wherein m is 0-3 and (CH)₂;

A is a hydrocarbyl group;

B is o-aminophenyl or hydroxyl group; and

Q is a halogen, hydrogen, or methyl,

to an animal in need of treatment wherein the activity of histone deacetylases is inhibited.