WO2011060268A2

WO2011060268A2 - Diagnosis and treatment of cancer using histone deacetylase inhibitors and radiolabeled metaiodobenzylguanidine

Info

Publication number: WO2011060268A2
Application number: PCT/US2010/056543
Authority: WO
Inventors: Karel Pacak; Lucia Martiniova; Frederieke M. Brouwers; Antonio Tito Fojo
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2009-11-13
Filing date: 2010-11-12
Publication date: 2011-05-19
Also published as: WO2011060268A3

Abstract

The present invention provides medicaments, their methods of preparation, as well as methods of diagnosis and imaging of mammalian tumors with [¹³¹I]-MIBG and/or [¹²³I]-MIBG. A method for treating a mammalian tumor which comprises administering to a mammal having a tumor, a composition comprising a histone deacetylase inhibitor, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about forty-eight hours, and followed by administering to the mammal a composition comprising an effective amount of [¹³¹I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof is also disclosed.

Description

DIAGNOSIS AND TREATMENT OF CANCER USING HISTONE DEACETYLASE INHIBITORS AND RADIOLABELED METAIODOBENZYLGUANIDINE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

61/260,991, filed on November 13, 2009, the entire contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Up to 36% of patients worldwide with pheochromocytoma (including both adrenal and extra-adrenal pheochromocytoma called paraganglioma) develop metastatic disease and have a 5-year survival rate of approximately 50% after diagnosis. Moreover, patients with metastatic pheochromocytoma exhibit excessive levels of circulating catecholamines, which results in an increased risk for strokes, cardiac arrhythmias, and hypertensive complications.

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Current treatments for malignant pheochromocytoma include targeted radiation using [ I]- metaiodobenzylguanidine ([ IJ-MIBG), cytotoxic chemotherapy, octreotide, tumor hemoembolization and, less frequently, radiofrequency ablation and cryotherapy. The success of these treatments is limited, and varies based on the sites and growth rate of metastatic lesions. [^I23I]-MIBG scintigraphy has been used for diagnosis and localization of malignant pheochromocytomas, and is effective for early detection of metastases and also as

131 a means of predetermining patients as potential candidates for radiotreatment with [ IJ- MIBG.

[0003] [¹³¹I]-MIBG is one of the most effective therapeutic options because it specifically targets chromaffin and pheochromocytoma cells. MIBG, a sympathomimetic amine analogue of guanethidine, is avidly taken up by chromaffin cells via the cell membrane norepinephrine transporter (NET). Similar to chromaffin cells, pheochromocytoma cells also express NET. As [¹³¹I] is covalently bound to MIBG, its uptake results in accumulation of [¹³¹I]-MIBG in the pheochromocytoma cells and their destruction by intracellular beta-radiation exposure. Unfortunately, only 30% of patients receiving [¹³¹I]-MIBG therapy show a positive tumor response. BRIEF SUMMARY OF THE INVENTION

[0004] In an embodiment, the present invention provides a method for treating a mammalian tumor which comprises administering to a mammal having a tumor, a

composition comprising an histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about twelve hours, followed by administering to the mammal a composition comprising an effective amount of [¹³¹I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0005] It is contemplated that in an embodiment of the present invention, the histone deacetylase inhibitors can be cyclic tetrapeptides, short chain fatty acids, hydroxamic acids and benzamides. In another embodiment, the histone deacetylase inhibitor is romidepsin or trichostatin A.

[0006] It is also contemplated that in an embodiment of the present invention, the methods of treatment disclosed herein are useful against many mammalian tumors, including pheochromocytomas, paragangliomas, neuroblastomas, ganglioneuromas, and intestinal carcinoids and carcinomas.

[0007] In another embodiment, the present invention provides a method for diagnosing a mammal for the presence of a mammalian tumor which comprises administering to a mammal a composition comprising a HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about twelve hours, followed by administering to the mammal a second composition comprising an effective amount of [¹³¹I]- MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof and detecting the presence of a mammalian tumor via beta-emission detection.

[0008] In a further embodiment, the present invention provides a method of radioimaging a mammalian tumor which comprises administering to a mammal a composition comprising a HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about twelve hours, followed by administering to the mammal a composition comprising an effective amount of [¹³¹I]-MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof; detecting the beta emission from the second composition within the mammal; and forming a high contrast image therefrom. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0009] Figure 1 illustrates dose and time dependent effects of romidepsin on MPC cell growth. Results are presented as mean ± SEM.

[0010] Figure 2 illustrates dose and time dependent effects of trichostatin A (TSA) on MPC cell growth. Results are presented as mean ± SEM.

[0011] Figure 3 depicts increased [ H]-NE uptake in MPC cells is shown after treatment with romidepsin (Fig. 3 A) and TSA (Fig. 3B) (* = p < 0.05: ** = p < 0.005, when compared to control (untreated cells)).

1 9^

[0012] Figure 4 is a series of three bar graphs depicting increased [ I]-MIBG uptake in MPC cells after treatment with romidepsin and TSA. The effect of the NET blockers

1 9^

desipramine (DMI) and reserpine (RES) on uptake time (10 minutes) of [ I]-MIBG after about 48 and about 72 hours of treatment in MPC cells with romidepsin and TSA is shown in Fig. 4A. Data are shown as percentage of [¹²³I]-MIBG uptake in treated cells compared to untreated control cells (baseline). The effect of the blockers DMI and RES on retention time (120 minutes) uptake of [¹²³I]-MIBG after 48 and 72 hours of treatment in MPC cells with romidepsin and TSA is shown in Fig. 4B. Figure 4C represents the uptake of [¹⁸F]-DA in MPC cells at 10 and 120 minutes. The effect on [^,8F]-DA was studied for 48 and 72 hours of romidepsin treatment, and the association of specific transporters with uptake of [ F]-DA was confirmed with the presence of a DMI blocker. DMI and RES were added 30 minutes prior to [¹²³I]-MIBG and [¹⁸F]-DA dosing. Results are presented as mean ± SEM. (NS = not significant, * = p O.05;** = p<0.005).

[0013] Figure 5 contains two graphs of the biodistribution of [ F]-DA (Fig. 5A) and [¹²³I]-MIBG (Fig. 5B) in liver lesions. The data show no correlation between a tumor's uptake of both radiopharmaceuticals and tumor size in either the controls or the treated groups. The tumor sizes are presented as mean ± SEM from at least three tumor samples.

[0014] Figure 6 depicts two graphs of dynamic PET acquisition which represents the pharmacokinetics of [¹⁸F]-DA in liver tumors, and in liver, in both pre-, and post-treatment animals, during a 60 minute imaging period (Figure 6A). Figure 6B is a graph summarizing whole PET imaging and showing [^I8F]-DA uptake as SUV_max (mean ± MEN) 6 days before, and 24 hours after treatment with a single intravenous dose of romidepsin (2.5 mg/kg), in

18

liver, pheochromocytoma liver metastases and muscle. The data show [ F]-DA uptake in the metastatic pheochromocytoma liver lesions is significantly increased after administration of romidepsin when compared to the uptake in the same lesions one week earlier on the pretreatment scan (p < 0.001).

[0015] Figure 7 shows representative pre- and post-treatment PET/MRI images of the same mouse. There was one week period between pre- and post-treatment. The tumor's

18 growth- over the one week is presented in MRI (left panels). The correlated uptake of [ F]- DA PET (right panel) is corresponding to the liver lesions imaged 24 hours after MRI.

[0016] Figure 8 is a bar graph depicting relative mRNA expression of NET in liver lesions samples after TSA treatment (n = 7) shown as fold induction over the expression in control untreated liver lesions (n = 6), and normalized expression of beta-actin.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present inventors contemplated that the poor response rate to current [¹³¹I]- MIBG therapy is most likely related to the under expression of NET that results in lower amounts of [¹³¹I]-MIBG concentrating within the tumor cells. Furthermore, the extremely elevated circulating catecholamines in the blood of pheochromocytoma patients compete with [¹³¹I]-MIBG for entry into the tumor cells through NET, thereby reducing [^13II]-MIBG uptake by the tumor. Because [¹²³I]-MIBG scintigraphy uses the same principle of being taken up by the NET of the cells and concentrating within a tumor, the present invention is also able to improve the diagnosis and identification of neuroendocrine metastatic tumors.

[0018] Histone deacetylase inhibitors (HDACi) are a new class of compounds that are known to arrest growth, induce differentiation and apoptosis in various cancer cell lines, and can inhibit tumor growth in animal models. The present inventors surprisingly found that HDACi can cause an increase in the amount of NET expressed in tumor

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(pheochromocytoma) cells. HDACi can be used to enhance uptake of [ I]-MIBG in scintigraphy and thus increase diagnosis and localization of possible tumors, as well as enhance [¹³¹I]-MIBG uptake in the tumor cells and increase the radiotherapeutic effect.

Moreover, it was found that if the tumor cells were exposed or pretreated with HDACi for a period of time of at least about 48 hours, preferably the cells were treated in a range of about 72 hours to about 96 hours, followed by administration of [ I]-MIBG, there was a significant increase in uptake of [¹³¹I]-MIBG in the tumor cells in vitro and in vivo, which improves the efficacy of the therapy.

[0019] In an embodiment, the present invention provides a method for treating a mammalian tumor which comprises administering to a mammal having a tumor, a composition comprising a HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about twelve hours, followed by administering to

131

the mammal a composition comprising an effective amount of [ IJ-MIBG or a

pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0020] It is contemplated that in an embodiment of the present invention, the histone deacetylase inhibitors can comprise cyclic tetrapeptides, short chain fatty acids, hydroxamic acids and benzamides. In another embodiment, the histone deacetylase inhibitor is romidepsin or trichostatin A.

[0021] It is also contemplated that in an embodiment of the present invention, the methods of treatment disclosed herein are useful against many mammalian tumors, including pheochromocytomas, paragangliomas, neuroblastomas, ganglioneuromas, intestinal carcinoids and carcinomas.

[0022] In another embodiment, the present invention provides a method for diagnosing a mammal for the presence of a mammalian tumor which comprises administering to a mammal a composition comprising a HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about twelve hours, followed by administering to the mammal a second composition comprising an effective amount of [ IJ- MIBG and/or [^I23I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof and detecting the presence of a mammalian tumor via beta-emission detection.

[0023] In a further embodiment, the present invention provides a method of radioimaging a mammalian tumor which comprises administering to a mammal a composition comprising a HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount for at least about twelve hours, followed by administering to the mammal a composition comprising an effective amount of [¹³¹I]-MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof; detecting the beta emission from the second composition within the mammal; and forming a high contrast image therefrom.

[0024] To carry out gene expression, a cell must control the coiling and uncoiling of DNA around histones. This is accomplished with the assistance of histone acetylases (HAT) which acetylate the lysine residues in core histones leading to a less compact and more transcriptionally active chromatin, and conversely the actions of histone deacetylases (HDAC) which remove the acetyl groups from the lysine residues leading to the formation of a condensed and transcriptionally silenced chromatin. [0025] HDAC inhibitors (HDACi) block this action, and can result in hyperacetylation of histones, therefore affecting gene expression. HDACi act exclusively on Class I and Class II HDACs by binding to the zinc containing catalytic domain of the HDACs. These HDACi fall into four general classes: Class I, hydroxamic acids, such as trichostatin A; Class II, cyclic tetrapeptides (such as trapoxin B) and the depsipeptides (romidepsin, spiruchostatin A); Class III, benzamides; and Class IV, short-chain fatty acids.

[0026] In an embodiment of the present invention, administration of the depsipeptide romidepsin upregulates the expression of the norepinephrine transporter protein (NET) in tumor cells. Other HDACis are suitable for use in the context of the methods of the present invention. For example, other suitable HDACis can include hydroxamic acids such as SAHA (trade name = Vorinostat®)(N-hydroxy-iV-phenyl-octanediamide), PXD101 (trade name = Belinostat®) (N-hydroxy-3-[3 [(phenylamino)sulfonyl]phenyl]-2-propenamide, and

LAQ824/LBH589 ((2E)-N-Hydroxy-3-[4-[[(2-hydroxyethyl)[2-(lH-indol-3- yl)ethyl]amino]methyl]phenyl]-2-propenamide); benzamides such as MS-275 (N-(2- aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide), CI994 (4- (Acetylamino)-N-(2-aminophenyl)benzamide), and MGCD0103. Other embodiments of the present invention include HDACis such as Apicidin [cyclo-L-(2-Amino-8-oxodecanoyl)-L- (N-methoxy-tryptophan)-L-isoleucyl-D-pipecolinyl], 5-Aza-2'-deoxycytidine, CAY10398 (4- (dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide), CAY10433 (N-phenyl-N'- (2-Aminophenyl)hexamethylenediamide), 6-Chloro-2,3,4,9-tetrahydro-lH-carbazole-l - carboxamide, HC Toxin (Cyclo (D-Prolyl-L-Alanyl-D-Alanyl-L-2-amino-9,10-epoxy-8- oxodecanoyl), ITSA1 (l-(2,4-Dichlorobenzoyl)-l H-benzotriazole), M344 (4-(Diethylamino)- N-[7-(hydroxyamino)-7-oxoheptyl] benzamide), MCI 293 (3 -(4-Toluoyl-l -methyl- 1H-2- pyrrolyl)-N-hydroxy-2-propenamide), Oxamflatin [(2E)-5-[3-[(phenylsufonyl) aminol phenyl]-pent-2-en-4-yn-hydroxamic acid], sodium butyrate, sodium 4-phenylbutyrate, and valproic acid.

[0027] It is known that current treatment options for malignant pheochromocytoma are

131

limited, and responses are often temporary. The success rate and response to [ IJ-MIBG, the most frequently applied therapeutic modality, depends on the dose and interval used to treat malignant pheochromocytoma. While response rates are increased with higher doses of [¹³¹I]-MIBG, higher doses are also associated with severe side effects. These side-effects are

131

the results of systemic beta-irradiation from the nonspecific uptake of [ I]-MIBG by normal 1 ^ 1

tissues. Moreover, high doses of [ IJ-MIBG can increase the long term risk of a second malignant neoplasm due to radiation exposure.

[0028] It was found that the therapeutic efficacy of [¹³¹I]-MIBG and [¹²³I]-MIBG could be increased, while at the same time decreasing their nonspecific uptake and treatment- associated damage in non-target tissues, by increasing the expression of NET in the tumor cells. NET in the cell membrane transports the [¹³¹I]-MIBG and [¹²³I]-MIBG across the plasma membrane into the tumor cells, such as pheochromocytoma cells. Increased expression of NET in tumor cells results in increased [^13II]-MIBG and [¹²³I]-MIBG uptake.

[0029] Accordingly, in an embodiment of the present invention, a method is provided for treating mammalian tumors in a mammal, comprising administering to the mammal having a tumor, a composition comprising a HDACi and/or its analog, derivative, isomer, metabolite, pharmaceutically acceptable salt, pharmaceutical product, hydrate, or N-oxide, or any combination thereof, in an amount effective to increase expression of NET in the cells of the tumor for at least about twelve hours. The time of exposure to HDACi must be sufficient so that the tumor cells have sufficient time to upregulate expression of NET proteins in significant quantity. Once sufficient exposure to HDACi has taken place, it is followed by administration of an effective amount of [¹³¹I]-MIBG to the mammal to cause tumor cell death.

[0030] It is also contemplated that the present invention can be used as a medicament for a range of disease conditions. Therefore, in an embodiment, the present invention provides a pharmaceutical composition comprising at least one a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, preferably for use as a medicament for inhibiting the growth of a mammalian tumor in a patient when administered in a period of time of about 12 hours to about 96 hours before treatment with a second composition comprising an effective amount of [¹³¹I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0031] With regard to the use of a medicament for inhibiting a mammalian tumor in a patient, the HDACi is selected from group consisting of cyclic tetrapeptides, short chain fatty acids, hydroxamic acids and benzamides. Preferably, the HDACi is romidepsin or trichostatin A. In addition, in an embodiment, the [^lJT]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof is administered after a period of time of about 48 to about 72 hours after the administration of at least one a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0032] With regard to the use of a medicament for inhibiting a mammalian tumor in a patient, the effective treatment amount of the HDACi or a pharmaceutically acceptable salt,

2 2 hydrate, or solvate thereof in the medicament, is from about 0.1 mg/m to about 24.9 mg/m

2 2

of the mammal, preferably from about 1 mg/m to about 15 mg/m , and more preferably from about 2 mg/m to about 3 mg/m .

[0033] With regard to the use of a medicament for inhibiting a mammalian tumor in a patient, the effective treatment amount of the HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof in the medicament, is from about 0.1 mg/kg to about 100 mg/kg, preferably from about 1 mg/kg to about 10 mg/kg, and more preferably from about 2 mg/kg to about 3 mg/kg of the mammal.

[0034] With regard to the use of a medicament for inhibiting a mammalian tumor in a patient, the mammalian tumor is selected from the group consisting of a pheochromocytoma, a paraganglioma, a neuroblastoma, a ganglioneuroma, an intestinal carcinoid, and an intestinal carcinoma.

[0035] In an embodiment, the present invention provides a pharmaceutical composition comprising a first composition comprising a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, preferably for use as a medicament for diagnosing a mammal for the presence of a mammalian tumor when administered in a period of time of about 12 hours to about 96 hours before treatment with a second composition comprising an effective amount of [¹³¹I]-MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and detecting the presence of a mammalian tumor via beta- emission detection.

[0036] In another embodiment, the present invention provides a pharmaceutical composition comprising a first composition comprising a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, preferably for use as a medicament for radioimaging a mammalian tumor when administered in a period of time of about 12 hours to about 96 hours before treatment with a second composition comprising an effective amount of [^I31I]-MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof, detecting the presence of a mammalian tumor via beta-emission detection, and forming a high contrast image therefrom.

[0037] It is also contemplated that the present invention provides for the use of a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for inhibiting the growth of a mammalian tumor in a patient when administered in a period of time of about 12 hours to about 96 hours before treatment with a second

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composition comprising an effective amount of [ I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof. The medicament of the present invention can be formulated to be intravenous, or oral. When the medicament is in an oral formulation, the form can be a sustained release formulation, and/or the medicament can be in a tablet, capsule, caplet, or lozenge form.

[0038] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of a disease in a mammal.

Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0039] As used herein, the term "detection," "imaging," or "radiodetection" means the use of certain properties of isotopes and the energetic particles emitted from radioactive material to diagnose or treat various medical conditions. In addition, the term "scintigraphy" means a diagnostic test in which a two-dimensional image of a body having a radiation source is obtained through the use of radioisotopes. A radioactive chemical is injected intravenously into the patient which then concentrates in the target cells or organ of interest. By placing a camera that senses radioactivity over the body, an image of the target cells or organ of interest can be created. The particles can be detected by suitable devices such as gamma cameras, positron emission tomography (PET) machines, single photon emission computed tomography (SPECT) machines and the like. The detection can be of either [ I]- MIBG and/or [¹²³I]-MIBG. When performing SPECT imaging, [¹²³I]-MIBG is preferably used.

[0040] In the present invention, in one embodiment, a suitable pharmaceutical composition is one in which the HDACi and [ I] -MIBG compositions used in the methods of the present invention are anchored on, or in, liposomes, and which can also contain a toxin, an anti-cancer drug or the like. The liposome used for anchoring the first and second compounds may be composed of a lipid bilayer. Alternatively, the liposome used may be composed of a multiple lipid layers or composed of a single lipid layer. Examples of the constituents of the liposome include phosphatidyl choline, cholesterol and phosphatidyl ethanolamine, and further include phosphatidic acid as a substance for imparting the liposome with electric charge. The ratio of those constituents is, for example, 0.3 to 1 mole, preferably 0.4 to 0.6 mole of cholesterol, 0.01 to 0.2 mole, preferably 0.02 to 0.1 mole of phosphatidyl ethanolamine, and about 0 to 0.4 mole, preferably about 0 to 0.15 mole of phosphatidic acid per 1 mole of phosphatidylcholine.

[0041] The methods of producing the liposome may be by any known conventional methods. For instance, they can be produced using a method in which a mixture of the lipids, from which a solvent has been removed, is emulsified by a homogenizer or the like, and then subjected to freeze-thawing to obtain a multilamellar liposome, followed by adjustment of pore size of the liposome appropriately by ultrasonication, high-speed homogenization, or pressure filtration through a membrane having uniform-size pores {Biochimica et Biophysica Acta., 812:793-801 (1985)). In an embodiment, it is contemplated that the liposomes have a particle size of about 30 nm to about 200 nm.

[0042] It is also contemplated that the present invention further includes HDACi derivatives. In one embodiment, the term "derivative" includes, but is not limited to, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. Methods of preparing these derivatives are known to a person skilled in the art. For example, ether derivatives are prepared by the coupling of the corresponding alcohols. Amide and ester derivatives are prepared from the corresponding carboxylic acid by a reaction with amines and alcohols, respectively.

[0043] In addition, this invention further includes hydrates of the HDACi compounds. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like. Hydrates of the HDACi compounds may be prepared by contacting the HDACi with water under suitable conditions to produce the hydrate of choice. [0044] As used herein, the invention provides a metabolite of the HDACi compounds. In one embodiment, the term "metabolite" refers to any substance produced from another substance by metabolism or a through a metabolic process of a living cell or organ.

[0045] In an embodiment, the compositions can include HDACi and [ IJ-MIBG and/or [¹²³I]-MIBG in conjunction with a carrier. The carrier is preferably a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of

administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0046] The choice of carrier will be determined in part by the particular HDACi used as well as by the particular method used to administer the HDACi. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. The following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary and are in no way limiting. More than one route can be used to administer the HDACi, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0047] Formulations suitable for parenteral administration of the compositions of the present invention include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0048] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. [0049] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0050] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the HDACi in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-iipophile balance (HLB) of, for example, from about 12 to about 17. The quantity of surfactant in such formulations will typically range from, for example, about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0051] Injectable formulations are in accordance with the present invention. The requirements for effective pharmaceutical carriers for injectable compositions are well- known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 14th ed., (2007)).

[0052] For purposes of the invention, the amount or dose of the HDACi and/or [¹³¹I]- MIBG administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject over a reasonable time frame. The dose will be determined by the efficacy of the particular HDACi and the condition of a human, as well as the body weight of a human to be treated. [0053] The dose of the HDACi and/or [¹³¹I]-MIBG and/or [^I23I]-MIBG also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular. Typically, the attending physician will decide the dosage of the HDACi and [^{13 I}I]-MIBG and/or [¹²³I]-MIBG with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, to be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the invention, the dose of the HDACi can be about 0.1 mg/m to about 24.9 mg/m to the subject being treated,

2 2

preferably from about 1 mg/m to about 15 mg/m . In an embodiment, the dose of HDACi administered is about 0.1 mg/kg to about 100 mg/kg, preferably from about 0.5 mg/kg to about 50 mg/kg, more preferably from about 1.0 mg/kg to about 10 mg/kg, and even more preferably from about 2 mg/kg to about 3 mg/kg. In an embodiment, the dose of HDACi administered is about 2.5 mg/kg in the mouse model, which is roughly the human equivalent of about 7.5 mg/m . Also by way of example and not intending to limit the invention, the

131 123

dose of the [^1JT]-MIBG and/or ['"I]-MIBG administered in nude mice can be about 1 μθϊ to about 100 μΟΊ, preferably from about 10 μΰΐ to about 50 μθ, more preferably from about 20 μα to about 30 μα. In an embodiment, the dose of [^I31I]-MIBG and/or [¹²³I]-MIBG administered is about 25 μθϊ, with a specific activity of about 2 mCi/0.08 mg.

[0054] The preparation of pharmaceutical compositions which contain the HDACi as an active component is well understood in the art, for example by mixing, granulating, or tablet-forming processes. In an embodiment, the HDACi ingredient is mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. For oral administration, the HDACis or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. For parenteral administration, the HDACis or their

physiologically tolerated derivatives, such as salts, esters, N-oxides, and the like are converted into a solution, suspension or emulsion, if desired, with the substances customary and suitable for this purpose, for example, solubilizers or other.

[0055] Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0056] For use in medicines, the salts of the HDACi will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable

pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

EXAMPLES

[0057] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0058] Two structurally different HDACi were tested. Romidepsin (a cyclic tetrapeptide) and TSA (a hydroxamic acid), were both tested in mouse pheochromocytoma (MPC) cells in vitro, and in a whole mouse model of metastatic pheochromocytoma in vivo.

[0059] RPMI 1640 medium, DMEM, Trypsin-EDTA, antibiotic, antimycotic, and N-2- hydroxyethylpiperazine-A'-2-ethane sulfonic acid (HEPES) buffer solution (IM) were purchased from Invitrogen-Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS, 500 ml) and donor horse serum (DHS, 500 ml) were obtained from Gemini Bio-Products

(Woodland, CA). Levo-2,5,6- H-norepinephrine was purchased from NEN Life Science Products (Boston. MA). Bovine serum albumin (BSA), dimethyl sulfoxide (DMSO) and all the components of the Krebs Ringer Glucose (H-KRG) buffer were purchased from Sigma Chemical Co. (St. Louis, MO). Phosphate buffered saline was obtained from Biofluids, Biosource International (Camarillo, CA), and Triton-X-100 (500 ml) from Fisher Scientific (Suwanee, GA). Sources for other materials are specified in the following paragraphs. Dr. A. T. Fojo (Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD, USA) kindly provided the romidepsin and TSA.

[0060] MPC cells (cell line 4/30/PRR), established from heterozygous neurofibromatosis knockout mice, and were kindly provided by Dr. A. Tischler (Powers, J.F., et al, Cell Tiss. Res., 302:309-320 (2000)). Cells were cultured in RPMI 1640 medium, supplemented with 10% DHS, 5% FBS, penicillin and streptomycin, and maintained at 37° C in a 5% C0₂ atmosphere. Cells from passages 26-38 were used in the experiments.

EXAMPLE 1

[0061] Cell Proliferation Assay. The cytotoxic effects of romidepsin and TSA were tested using the XTT-assay (cell proliferation kit II, Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, IN). MPC cells were seeded in 96-well flat-bottom plates (50,000 cells/well) and incubated with increasing concentrations of romidepsin and TSA at 37° C for 48 and 72 hours. Romidepsin and TSA were diluted in medium. The XTT labeling mixture was then added to the plates, and cells were incubated for 18 additional hours. After incubation, spectrophotometric absorbance was measured as described by a microplate reader (Bio-Rad Laboratories, Philadelphia, PA). All experiments were performed in octuplicate.

EXAMPLE 2

[0062] Uptake experiments in vitro with [³H]-norepinephrine. The uptake of

norepinephrine was determined in MPC cells using [³H] -norepinephrine ([³H]-NE) by modification of the protocol described by Jaques et al. (Jaques S., et al., Mol. Pharmacol., 26:539-546 (1984)). Due to the long half-life of ³H (12.3 years), initial in vitro experiments were performed using [³H]-NE. MPC cells, grown in 24-well plates (100,000 cells/well), were treated with increasing concentrations of romidepsin (0.001-10 ng/ml) or TSA (6.25- 100 ng/ml) for about 48 or about 72 hours at 37° C. After treatment, cells were washed three times with 0.5 ml H-KRG buffer (H-KRG: 125 mM NaCl, 4.8 mM KC1, 2 6 mM CaCl₂, 1.2 mM MgS0₄, 5.6 mM glucose, 25 mM HEPES, 1 mM ascorbic acid, at a pH of 7.35), followed by a 10 minute preincubation in H-KRG buffer (0.5 ml/well at 37° C). Next, [³H]- NE (25 nM/well, specific activity of about 40-80 Ci/mmol) was added into medium and cells were incubated at 37° C for 10 min ("uptake"). After incubation, [³H]-NE uptake was stopped by rapidly chilling the plates on ice, and cells were washed twice with PBS and supplemented with 0.1% albumin at 4° C. Cells were then lysed with 0.5 ml Triton-X 0.1% and aliquots of the cell lysates were transferred into scintillation vials. After addition of the Biosafe-II scintillation cocktail (Research Products International, Mount Prospect, IL), cell- associated beta radiation was counted in a beta-counter (LS 60000 IC, Beckman Coulter Inc., Brea, CA). EXAMPLE 3

[0063] Uptake and retention experiments in vitro with [¹²³I]-MIBG and [¹⁸F]-DA. The [ I]-MIBG and [ F]-DA uptake studies, the treatment of cells, washing steps, and incubations were performed following the same protocol used for [ H]-NE. MPC cells were grown in 24-well plates (100,000 cells/well) and treated with romidepsin (0.5 ng/ml) or TSA (12.5 ng/ml) for about 48 or about 72 hours, at 37° C. [¹²³I]-MIBG (0.6 to 0.7 μθί/ιηΐ, specific activity of about 2 mCi/0.08 mg), [¹⁸F]-DA (0.6 to 0.7 μα/ml, specific activity of either about 20 mCi/3.23 mg or about 17.31 mCi/2.49 mg) was added to the plates, which were incubated at 37° C for 10 minutes ("uptake") and 120 minutes ("retention uptake"). After incubation, [¹²³I]-MIBG uptake was stopped by rapidly chilling the plates on ice, and cells were washed twice with PBS supplemented with 0.1% albumin at 4° C. Then, the incubation medium was collected, and the cells were washed with cold PBS, trypsinized, and the cell-associated gamma radiation was measured using a gamma-counter (1480 Wizard 3, Automatic Gamma Counter, Perkin-Elmer, Waltham, MA). In a subset of experiments, uptake studies with romidepsin and TSA treatment were carried out in the absence or presence of 1 μΜ DMI to block catecholamine neuronal uptake, and 10 μΜ reserpine to block vesicular translocation of catecholamines. All experiments were performed in quadruplicate.

EXAMPLE 4

[0064] Quantification of NET expression. Expression of NET was quantified by quantitative PCR as previously described by others (Muiphy et al., Biochemistry, 29: 10351- 10356 (1990)). Total RNA was extracted from MPC cells using the Trizol reagent (Life Technologies, Inc. Rockville, MD). The following primers were used: 5'-primer 5'- GC ATC AATGCCTACTTGC AC-3 ' (SEQ ID NO: 1) and 3'-primer 5'- AGGATAAACACAAGCCCAGC-3 ' (SEQ ID NO: 2), yielding a 301 base pair product. The PCR settings were as follows: 7 minutes at 94° C, 20 thermocycles of 240 seconds (75 seconds at 94° C, 75 seconds at 55° C and 90 seconds at 72° C), 10 thermocycles of 270 seconds (75 seconds at 94° C, 75 seconds at 55° C and 120 seconds at 72° C), followed by 10 minutes at 72° C. EXAMPLE 5

[0065] Protein collection and analysis. For these experiments, cells were incubated with 0, 0.25, 0.5 or 0.75 ng/ml of romidepsin, and 0, 6.25, 12.5 or 25 ng/ml of TSA for 72 hours in 25 ml flasks. After two washes with PBS, cells were pelleted and lysed. Cell lysates were added to the loading buffer (1 :1 GTC/PBS, Invitrogen/Cellgro). The samples were serially diluted 1 :2 and dilutions were loaded into a 96-well vacuum manifold (Bio-Rad) with two sheets Protran® membrane (Whatman, Piscataway, NJ), with a vacuum applied slowly. The membrane was removed and washed twice in tris buffered saline (TBS) before being probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at 1 :500 (American Research Products). After antibody hybridization, the membrane was washed three times in Tween® tris buffered saline (TTBS). The blot was then hybridized with horseradish peroxidase linked anti-mouse Ig whole antibody at 1 : 1000 (Amersham, GE Healthcare Inc., Piscataway, NJ) and imaged using ECL western blotting kit (Amersham). Next, the membrane was stripped using Re-Blot Plus Mild (Millipore Inc., Billerica, MA) for ten minutes and subsequently, it was probed for anti-acetyl histone H3 (1 :2200) and again washed three times in TTBS, followed by horseradish peroxidase linked anti -rabbit Ig whole antibody at 1 : 1000

(Amersham), and imaged as before. Densitometry was performed on films using the IPLab gel software (Signal Analytics Corp., Vienna, VA).

EXAMPLE 6

[0066] Ultrastructural Studies. The effect of treatment with either 0.5 ng/ml romidepsin, or 12.5 ng/ml TSA, on MPC cell morphology and ultrastructure was examined by electron microscopy. After 3 washes with PBS, cells were double-fixed in PBS-buffered

glutaraldehyde (2.5%) and osmium tetroxide (0.5%), dehydrated, embedded into Spurr's epoxy resin, and coded to insure unbiased assessment. Ultrathin sections (90 nm) were made and double-stained with uranyl acetate and lead citrate and viewed in a Philips CM 10 transmission electron microscope (Philips Electronics N.V., Amsterdam, Netherlands).

EXAMPLE 7

[0067] In vivo studies with an animal model. Establishment of the mouse model of metastatic pheochromocytoma used in this study was previously described in detail

(Martiniova, L. et al., Clin. Exp. Metastasis, 26:239-250 (2009)). Briefly, adult six to eight weeks, (20-22 g) female athymic nude mice (NCr-nu, Taconic, Germantown, NY) were injected by tail vein with lxlO⁶ MPC cells (MPC 4/30 PRR). MPC cells were mixed with 100 μΐ of PBS (Sigma Chemicals Co., St. Louis) and held at room temperature during processing. Mice were anesthetized using isoflurane/0₂ (1.5-5% v/v) before all imaging and treatment procedures. All animal studies were conducted according to the National Institute of Health Guide for the Care and Use of Animals, under an approved protocol from the National Institutes of Health Institutional Animal Care and Use Committee.

EXAMPLE 8

[0068] In vivo magnetic resonance imaging (MRI). Magnetic resonance images were obtained with a 3 Tesla MRI scanner (Intera, Philips Medical System, Best, Netherlands) using a dedicated 40 mm inner diameter solenoid coil (Philips). Localization and monitoring of liver pheochromocytoma lesions were carried out as previously described in (Martiniova, L., et al., J Magn. Reson. Imaging, 29:685-691 (2009)). Briefly, anesthetized animals in the prone position, respiratory triggered T₂-weighted acquisition with the following parameters were acquired: FOV 8.0 x 8.0 x 2.0 cm³, data matrix 512 x 512, 40 slices, TE/TR 6S/4500 ms, flip angle 90°, slice thickness 0.5 mm. 0.156 x 0 156 mm² in-plane resolution, scan line of 5-7 minutes, for two signal averages depending on the respiratory rate. No contrast agent was used for MRI. Animals were scanned 4-5 weeks after the injection of MPC cells to determine liver tumors size.

EXAMPLE 9

[0069] [¹²³I]-MIBG and [¹⁸F]-DA biodistribution studies. Romidepsin dose treatment was followed by the protocol described by Goldsmith et al. (Goldsmith M.E., et al., Mol. Cancer Ther., 6:496-505 (2007)), with a small modification. In the experiments, xenografted mice were treated with romidepsin at a dose of about 3.6 mg/kg, however, nude mice with liver metastases in presented model did not tolerate that dose. Thus, a lower, single dose of romidepsin was administered in a concentration of about 2.5 mg/kg to evaluate accumulation and storage in the tumors. The same concentration was chosen for TSA. Both romidepsin and TSA were resuspended in saline and administered intravenously (/.v.) with an injection volume of approximately 150 μΐ at a rate of 10 μΐ per min. Scans were taken at baseline and 24 hours after drug administration. [0070] For in vivo biodistribution studies, anesthetized nude mice were injected i.v.

through the lateral tail vein with a total volume of 150 μΐ saline solution containing about 2.5 mg/kg of TSA (n = 3) by infusion over approximately 15 minutes. TSA was injected twice, 24 hours and 2 hours before administration of 25-27 μθ of [^I23I]-MIBG. Control untreated mice (n = 4) were injected i.v. through the lateral tail vein with a total volume of 150 μΐ saline solution and vehicle, and eventually were injected with the same dose of [^I23I]-MIBG. Each group was sacrificed by cervical dislocation at approximately 120 minutes post injection of

123

[ I]-MIBG. A single dose of romidepsin (about 2.5 mg/kg) was injected into mice (n = 7) 24 hours before about 50-60 μθϊ of [¹⁸F]-DA administration. Control mice (n = 7) were injected with the same dose [¹⁸F]-DA. Both groups were sacrificed 60 minutes post tracer injection. Samples of liver lesions, normal liver were collected and weighed. The [¹²³I]- MIBG radioactive content of the liver tumors and tissues was assayed using an automatic gamma counter (Perkin Elmer, model 1480 Wallac Wizard, Rockwall, TX). Standards of 1 : 10 of the injected dose (ID) were prepared and counted along with all samples.

Background counts were subtracted from the reported ¹²³I counts per minute (CPM). The injected counts were determined from the standard counts and the quantitative data, expressed as standardized uptake value (SUV) determined as described previously (Green M.V., et al., Comput. Med. Imaging Graph., 25:79-86 (2001)).

EXAMPLE 10

18

[0071] [ F]-DA positron emission tomography imaging. Positron emission tomography is a noninvasive technique which allows the monitoring of tumors longitudinally. The procedure for romidepsin pretreatment evaluation consisted of two [¹⁸F]-DA scans (2.5 mg/kg. n = 4 mice), one before treatment, as a baseline scan, and one 24 hours after treatment with romidepsin on the same mice. Animal PET scans were performed with the Advanced Technology Laboratory Animal Scanner (ATLAS) (Seidel J., IEEE Trans. Nuc. Set,

50: 1347-1350 (2003)), which has a transverse field-of-view (FOV) of 6.8 cm and an axial FOV of 2 cm. PET images were reconstructed by 2D-ordered-subsct expectation

maximization (2D OSEM) algorithm (5 iterations and 16 subsets), achieving a 1.5-mm full width at half maximum (FWHM) resolution at the center (Toyama H., et al., J. Nuc. Med., 45:1398-1405 (2004)). The reconstructed voxel size was 0.56 x 0.56 x 1.125 mm³. No correction was applied for attenuation or scatter. Dynamic data acquisition determining

18

pharmacokinetic of [ F]-DA in liver tumors and liver parenchyma started about 1 minute after radiotracer injection. Scanning parameters were set for one frame/ 10 minutes up to 6 frames. Whole body data acquisitions (2 bed positions, each 10 minutes) started

consecutively after the dynamic acquisition. Whole body acquisitions (achieving 2 x 2 cm = 4 cm of the field of view) included images of the lungs through the kidneys were acquired after administration of 50-60 μθϊ of [¹⁸F]-DA.

[0072] Each liver lesion was analyzed individually for the maximal uptake based on the most active voxel cluster located within the region of interest (ROI). This is equivalent to the maximum standardized uptake value (SUV_max) used in clinical PET studies. If liver tumors are smaller (in any dimension) than approximately 2.5 x FWHM spatial resolution of the PET scanner, a distortion called partial volume effect occurs. Therefore, only those liver lesions were analyzed if larger than 4 mm in diameter as obtained by MRI. This is the minimum size required to exclude partial volume effects in small animal PET, when monitoring lesions for potential physiological changes. The ¹⁸F activity concentration of the radionuclide was expresses as tumor-to-liver ratio (TLR), obtained by comparing uptake in the liver lesions with that in the liver parenchyma.

[0073] Results herein are presented as the mean ± SEM from a minimum of three experiments for both in vitro and in vivo experiments. Before performing any statistical test, all data were tested for normal distribution and equal variance. Statistical differences between groups of data were assessed by ANOVA, followed by Student-Neuman-Keuls test for group comparison. The level of statistical significance was set at p < 0.05.

[0074] Treatment with romidepsin or TSA induced a dose-dependent decrease m MPC cell proliferation. Growth inhibition curves after about 48 and about 72 hours of exposure to the drugs are shown in Figs. 1 and 2. After about a 72-hour exposure, the 50% inhibitory concentrations (IC₅₀) were about 1.56 ng/ml (from studied range 0.001 ng/ml-1000 ng/ml) for romidepsin and about 50 ng/ml (from studied range 1 ng/ml- 100 ng/ml) for TSA.

[0075] A significant dose and time-dependent increase in the specific [³H]-NE uptake was observed in MPC cells treated with romidepsin concentrations of about 0.25-0.5 ng/ml, and TSA concentrations of about 6.25-12.5 ng/ml. This increase was followed by a decrease at the highest concentrations of both romidepsin and TSA (Figs. 3 A and 3B). The maximal

• * 3

increase m [ H]-NE uptake occurred in MPC cells after about 72 hours of treatment with around 0.5 ng/ml of romidepsin and around 12.5 ng/ml of TSA, respectively. In further experiments concentrations of about 0.5 ng/ml romidepsin and about 12.5 ng/ml TSA were used. [0076] The maximal increase of [ I]-MIBG uptake in MPC cells occurred after 72 hours after treatment with romidepsin (0.5 ng/ml) and TSA (12.5 ng/ml), with the uptake time of about 10 minutes, and the retention time of about 120 minutes (Figs. 4A and 4B). The uptake of [ IJ-MIBG in MPC cells was approximately 3.6 times higher than in the control group at 10 minutes and about 4.4 times higher around 120 minutes after 72 hours of treatment with TSA. Similarly, for romidepsin the uptake of [ I]-MIBG was approximately 1.5 times higher than in the control group at 10 minutes and about 2.2 times higher at about 120 minutes.

[0077] In order to find out whether the increase of [¹²³I]-MIBG uptake was due to inhibition of transport over the cellular membrane via NET, or due to impaired granular storage, a comparison of the effect of DMI on cellular [^I23I]-MIBG uptake and retention, as well as a comparison of the effect of reserpine on storage of [¹²³I]-MIBG in vesicles, was performed. DMI inhibited the entry of [^!23I]-MIBG into both untreated and treated MPC cells. The inhibitory effect was more pronounced at 10 minutes than at 120 minutes (Figs. 4 A. and 4B). A similar inhibitory effect was observed with the vesicular monoamine transporter (VMAT) blocker reserpine, at 120 minutes in untreated cells, and at about 10 minutes and about 120 minutes in cells treated with TSA (Figs. 4A. and 4B).

[0078] Similarly, increased accumulation of [ F]-DA was observed in MPC cells after treatment with romidepsin for about 48 and about 72 hours (Fig. 2C). DMI significantly inhibited the entry of [¹⁸F]-DA into MPC cells (Fig. 4C).

[0079] Quantitative PCR data showed an approximate 1.5-2-fold increase in NET expression after about 72 hours of treatment with about 0.5 ng/ml of romidepsin and about 12.5 ng/ml TSA, in comparison to untreated cells (data not shown).

[0080] As expected, blot analysis showed increased acetylation of histone H3 in MPC cells by the same doses of romidepsin and TSA that had been proven to influence

proliferation and differentiation, [ H]-NE and [ IJ-MIBG uptake, and neurosecretory granules accumulation in these cells (data not shown).

[0081] Romidepsin and TSA-treated cells showed no ultrastructural evidence of increased apoptosis (i.e., aggregation of chromatin into dense sharply delineated masses, condensed cytoplasm, and apoptotic bodies) compared to untreated MPC cells (data not shown).

[0082] MRI Imaging. Respiratory triggered T₂-weighted MRI detected multiple liver lesions in an animal model were performed. Liver lesions of the size of 4 mm in diameter were found five weeks after injection of MPC cells. Lesions at this size were suitable for PET imaging. Moreover, MRI was used to follow the tumor over weeks until the liver lesions would develop in suitable size for subsequent functional imaging. Also,

determinations of the tumor growth of individual lesions and comparisons of the radiotracer uptake with the size of the lesions were made as well.

[0083] Tumor bearing, nude mice were sacrificed at 120 minutes post [¹²³I]-MIBG injection. Liver lesions of various sizes were taken out (from about 2 mm up to about 8 mm

123

in diameter) to determine whether there existed a correlation between [ IJ-MIBG uptake and tumor size. In summary, the radioactive concentration of [ IJ-MIBG in liver metastases (in SUV units) after treatment with TSA was about 2.08 (± 0.287), compared to about 0.87 (± 0.123) in controls (p < 0.001). The corresponding TLR values were around 3.09, and around

1.82 respectively. Similar results were observed for the [ F]-DA biodistribution (in SUV), in mice treated with romidepsin (3.522 ± 0.329 vs. 0.926 ± 0.464, p < 0 001). The TLR value was significantly higher in the treated group versus the control group (4.402 vs. 1.12 respectively). Figure 5 illustrates that there is no apparent correlation between a tumor's uptake of both radiopharmaceuticals [ F]-DA and [ IJ-MIBG, and that tumor's size in either the control or treated groups. The increase of the uptake in liver tumors after [ FJ-DA and [ 123 IJ-MIBG i *njection is independent of their size, and thus, can only be as a result of increased NET expression and retention in storage vesicles in the treated cells. These results are congruent with the in vitro findings.

[0084] The effect of romidepsin in the same mice was also evaluated by PET imaging.

Liver lesions were measured by MRI one day before [ FJ-DA administration, both pre- and post-romidepsin treatment. Representative dynamic PET images, acquired over 60 minutes after injection of [ FJ-DA, showed increased tracer accumulation in post treatment scans compared to pretreatment scans (Fig. 6A). Whole body PET images confirmed the increased accumulation of [ FJ-DA in pheochromocytoma liver metastases in all 4 mice, after romidepsin treatment. Uptake of [ FJ-DA was higher in liver lesions, than in normal liver parenchyma, as shown in pretreatment scans (liver lesions SUV_max 1.259 ± 0.357 vs. liver parenchyma SUV_max 0.66 ± 0.203. p O.001). However, [ FJ-DA uptake was significantly higher in liver lesions, when compared to liver parenchyma in the same mice (3.8 ± 1.44 vs. 0.60 ± 0 11, p < 0.001) after treatment with romidepsin (Fig. 6B). Representative pre- and post-treatment PET/MRI images of the same mouse are presented in Figure 7. The grey scale indicates SUV_max, values in PET images. TLR values were about 1.9 for the pretreatment scan, and about 6.3 for post-treatment accumulation. These results were consistent with the increased [ⁱ⁸F]-DA activity concentration detected in liver lesions.

[0085] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0086] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0087] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A pharmaceutical composition comprising at least one a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, preferably for use as a medicament for inhibiting the growth of a mammalian tumor in a patient when administered in a period of time of about 12 hours to about 96 hours before treatment with a second composition comprising an effective amount of [^1JT]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

2. The pharmaceutical composition of claim 1 , wherein the HDACi is selected from group consisting of cyclic tetrapeptides, short chain fatty acids, hydroxamic acids and benzamides.

3. The pharmaceutical composition of either of claims 1 or 2, wherein the HDACi is romidepsin or trichostatin A.

4. The pharmaceutical composition of any of claims 1-3, wherein the second composition is administered after a period of time of about 48 to about 72 hours after the administration of the pharmaceutical composition of claim 1.

5. The pharmaceutical composition of any of claims 1-4, wherein the effective treatment amount of the HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, is from about 0.1 mg/m² to about 24.9 mg/m² of the mammal, preferably from about 1 mg/m² to about 15 mg/m², and more preferably from about 2 mg/m² to about 3 mg/m².

6. The pharmaceutical composition of any of claims 1-4, wherein the effective treatment amount of the HDACi or a pharmaceutically acceptable salt, hydrate, or solvate thereof, is from about 0.1 mg/kg to about 100 mg/kg, preferably from about 1 mg/kg to about 10 mg/kg, and more preferably from about 2 mg/kg to about 3 mg/kg of the mammal.

7. The pharmaceutical composition of any of claims 1-6, wherein the mammalian tumor is selected from the group consisting of a pheochromocytoma, a paraganglioma, a neuroblastoma, a ganglioneuroma, an intestinal carcinoid, and an intestinal carcinoma.

8. A pharmaceutical composition comprising a first composition comprising a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, preferably for use as a medicament for diagnosing a mammal for the presence of a mammalian tumor when administered in a period of time of about 12 hours to about 96 hours

131 before treatment with a second composition comprising an effective amount of [ I]-MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and detecting the presence of a mammalian tumor via beta-emission detection.

9. A pharmaceutical composition comprising a first composition comprising a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, in an amount effective for use in a medicament, preferably for use as a medicament for radioimaging a mammalian tumor when administered in a period of time of about 12 hours to about 96 hours before treatment with a second composition comprising an effective amount of [¹³¹I]-MIBG and/or [¹²³I]-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof, detecting the presence of a mammalian tumor via beta-emission detection, and forming a high contrast image therefrom.

10. Use of a histone deacetylase inhibitor (HDACi) or a pharmaceutically acceptable salt, hydrate, or solvate thereof, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for inhibiting the growth of a mammalian tumor in a patient when administered in a period of time of about 12 hours to about 96 hours before

131

treatment with a second composition comprising an effective amount of [ IJ-MIBG or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

1 1. The use of claim 10, wherein the medicament is formulated for intravenous administration.

12. The use of claim 10, wherein the medicament is formulated for oral administration.

13. The use of claim 12, wherein the medicament is an immediate-release or a sustained release formulation.

14. The use of claim 12 or 13, wherein the medicament is a tablet, capsule, caplet, or lozenge.