WO2008098351A1

WO2008098351A1 - Treatment of d-cyclin mediated proliferative diseases and hemotological malignancies

Info

Publication number: WO2008098351A1
Application number: PCT/CA2008/000260
Authority: WO
Inventors: Aaron D. Schimmer; Xinliang Mao; Keith Stewart
Original assignee: University Health Network
Priority date: 2007-02-14
Filing date: 2008-02-12
Publication date: 2008-08-21

Abstract

The treatment of proliferative diseases involving D-cyclin and/or hematological malignancy using compounds of Formula ( I ), as well as methods for identifying D-cyclin modulators are disclosed. Wherein R1 is selected from H, C1-4alkyl, C(O)OC1-4 alkyl, C(O)C1-4 alkyl and C(O)NH C1-4 alkyl, R2 is selected from H and C1-4 alkyl, R3 is selected from H and C1-4 alkyl, or R2 and R3 are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring, R4 and R5 are independently selected from H, halogen, hydroxy, C1-4 alkyl, fluoro-substituted C1-4 alkyl and C1-4 alkoxy, and X is selected from C and N.

Description

B&P Reference 10723-182 TITLE: Treatment of Proliferative Diseases

FIELD OF THE APPLICATION The disclosure relates to methods and compositions for the treatment of proliferative diseases involving increased D-cyclin expression and/or hematological malignancies in a subject.

BACKGROUND OF THE APPLICATION Acute myeloid leukemia (AML) and multiple myeloma (MM) are malignant diseases resulting in the proliferation of abnormal cells of myeloid and lymphoid origin, respectively. Both diseases are characterized by poor responses to standard therapies. For example, elderly patients with either AML or myeloma and poor risk cytogenetics have a median survival of less than one year. Thus, for these patients and those with relapsed refractory disease novel therapies are needed. As many of these patients are frail, therapies that achieve an anti-myeloma or leukemia effect without significant toxicity are highly desirable.

Mitogenic signals stimulate progression of the cell cycle and the transition of cells from G₀ to Gi and from Gi to S-phase, thereby promoting cell growth and proliferation. Proper control of the cell cycle is critical to achieving the optimal amount of cell division and proliferation. In malignancies such as MM, the control of cell cycle is dysregulated leading to increased plasma cell proliferation (2, 4). Progression from Gi to S phase is controlled by the Rb tumor suppressor gene and the D-type cyclins (4-6).

Cyclin D1 , cyclin D2, and cyclin D3 are homologous proteins that promote cell cycle progression by forming complexes with cyclin dependent kinases (CDKs) that promote the phosphorylation of Rb (7). Over-expression of cyclins leads to increased cell proliferation (1 , 2, 8) and chemoresistance (9-11). In almost all cases of MM, at least one of the three cyclins is over- expressed (2). In approximately 50% of cases, expression of cyclin D2 is dysregulated (2, 3). Dysregulation of cyclin D2 can result from over- expression of transactivators such as the transcription factor c-maf (12). Alternatively, dysregulation of signaling pathways such as the FGFR (fibroblast growth factor receptor) kinase pathway leads to up-regulation of cyclin D2 (3, 4, 11). In malignant cells, multiple studies (including (13-15)) have demonstrated that decreasing D-cyclins arrests cells in the Gi phase of the cell cycle and induces apoptosis.

Treatments for various cancers are known in the art, although it is difficult if not impossible to predict ab initio the effect a particular combination of drugs may have. US2004/0072824A1 relates to methods of treating and managing cancer comprising the administration of an anti-histaminic agent or structurally/functionally related compound. WO1994/018961 describes chemotherapeutic treatment of cancer cells is improved by first administering a compound which inhibits normal cell proliferation while promoting malignant cell proliferation, specifically a potent antagonist selective for intracellular histamine receptors, in an amount sufficient to inhibit the binding of intracellular histamine to the receptors in normal and malignant cells. An enhanced toxic effect on the cancer cells from the chemotherapeutic agent is obtained while any adverse effect of the chemotherapeutic agent on normal cells is inhibited. US4,771 ,056 describes cell proliferation may be promoted by serotonin. Specific inhibitors of serotonin uptake were examined for their effects on cell proliferation. The agents suppressed cell division in dimethylhydrazine induced colonic tumors in rats.

SUMMARY OF THE APPLICATION

The present application provides a novel treatment for proliferative diseases involving increased expression of D-cyclins and/or hematological malignancies, such as leukemias including acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL), and multiple myeloma (MM) using a compound selected from a compound of Formula I, and salts, solvates and prodrugs thereof, a compound of Formula I being:

wherein

^^ is a single or double bond, R¹ is selected from H, C_1-4alkyl, CCOJOC^alkyl, C(O)Ci-₄alkyl and C(O)NHCi-

₄alkyl;

R² is selected from H and

R³ is selected from H and Ci^alkyl; or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring;

R⁴ and R⁵ are independently selected from H, halogen, hydroxy, Ci^alkyl, fluoro-substituted

and

X is selected from C and N.

One aspect of the present application is a method of treating a proliferative disease involving increased D-cyclin expression comprising administering, to a subject in need of such treatment, an effective amount of compound selected from a compound of Formula I, and salts, solvates and prodrugs thereof.

Another aspect of the application is the use of a compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof, for the treatment of a proliferative disease involving increased D-cyclin expression. A further aspect of the application is a use of compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof, in the preparation of a medicament for the treatment of a proliferative disease involving increased D-cyclin expression.

A further aspect of the application is a method for the treatment of a hematological malignancy comprising administering, to a subject in need of such treatment, an effective amount of compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof. In certain embodiments the hematological malignancy is leukemia, such as acute myeloid leukemia or acute lymphocytic leukemia, in other embodiments the hematological malignancy is multiple myeloma.

Yet another aspect of the application is the use of a compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof, for the treatment of a hematological malignancy. In one embodiment the hematological malignancy is acute myeloid leukemia, acute lymphocytic leukemia or multiple myeloma.

Another aspect of the application is the use of a compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof, in the preparation of a medicament for treatment of a hematological malignancy. In one embodiment, the hematological malignancy is acute myeloid leukaemia, acute lymphocytic leukemia or multiple myeloma.

In an additional aspect of the application the effective amount of the compound of Formula I, and/or a pharmaceutically acceptable salt, solvate or prodrug thereof, is within the range of about 0.1 to about 200 mg/kg body weight, suitably in the range of about 0.1 to about 100 mg/kg body weight, about 0.1 to about 80 mg/kg body weight, about 0.1 to about 40 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 0.1 to about 2 mg/kg body weight, or about 0.5 to about 2 mg/kg body weight.

In other embodiments, the effective amount of the compound of Formula I, and/or a pharmaceutically acceptable salt, solvate or prodrug thereof, is within the range of about 2 to about 2000 mg, about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg, or about 35 to about 50 mg daily dosage. In further embodiments the methods or uses comprise chronic administration, wherein the effective amount of the compound of Formula I and/or a pharmaceutically acceptable salt, solvate or prodrug thereof, is within the range of about 2 to about 2000 mg, about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg, or about 35 to about 50 mg and administration or use is one or more times daily for 1 to 2 weeks, 2 to 4 weeks, and/or more than 4 weeks.

In other embodiments the methods comprise administering a pharmaceutical composition described herein. In yet other embodiments, the uses comprise use of a pharmaceutical composition described herein. A further aspect of the application is a pharmaceutical composition for the treatment of proliferative diseases involving increased expression of D-cyclins and/or hematological comprising a compound selected from a compound of Formula I and a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier in a dosage form. In a further embodiment, the pharmaceutical composition is formulated for oral administration or injection.

A further aspect of the application is a pharmaceutical composition wherein a solid dosage form contains from about 2 to about 2000 mg of a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate or prodrug thereof, suitably from about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg or about 35 to about 50 mg of the compound.

A further aspect of the application is a pharmaceutical composition wherein the dosage form is a liquid dosage form that contains from about 2 to about 2000 mg a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate or prodrug thereof, suitably from about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg, about 35 to about 50 mg, of the compound. A further aspect of the application is a pharmaceutical composition comprising a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier in unit dosage form in an amount suitable to provide about 0.1 to about 200 mg /kg body weight, suitably about 0.1 to about 100 mg/kg body weight, about 0.1 to about 80 mg/kg body weight, about 0.1 to about 40 mg/kg/body weight, about 0.1 to about 20 mg/kg body weight about 0.1 to about 2 mg/kg body weight or about 0.5 to about 2 mg /kg body weight of the compound, formulated into a solid oral dosage form, a liquid dosage form, or an injectable dosage form.

A further aspect of the application is a composition formulated as an oral dosage form selected from enteric coated tablets, caplets, gelcaps, and capsules, comprising from about 5 to less than about 2000 mg, suitably from about 10 to about 100 mg, about 30 to about 60 mg, or about 35 to about 50 mg, of a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate or prodrug thereof and a pharmaceutically acceptable carrier. Suitably each tablet, caplet, gelcap or capsule comprises about 2 to 2000 mg, suitably about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg or about 35 to about 50 mg of the compound.

A further aspect of the application is a commercial package comprising a composition according to the present application, and associated therewith instructions for the use thereof for treatment of proliferative diseases involving increased expression of D-cyclins or hematological malignancies such as leukemias including acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL), and multiple myeloma (MM).

Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the application are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the application will now be described in relation to the drawings in which: Figure 1 is a schematic representation of a high throughput assay to identify c-maf dependent and independent inhibitors of cyclin D2 transactivation. Figure 2 is a graph demonstrating the identification of small-molecule inhibitors of the cyclin D2 promoter.

Figure 3 is a work flow diagram of the screen for inhibitors of the cyclin D2 promoter. Figure 4 is a series of graphs showing characterization of cyclin D2 promoter inhibitors. A) A graph colormetrically illustrating the clustering and characterization of small-molecule inhibitors of the cyclin D2 promoter B) A graph illustrating inhibition of the cyclin D2 promoter with increasing concentrations of cyproheptadine. Figure 5 is a series of scans of immunoblots demonstrating that cyproheptadine reduces levels of cyclin D1, D2, and D3 in MM and AML cell lines. A) Effect of cyproheptadine (20 μM) on cyclin D1 , D2, and D3 in KMS12, LP1 , My5, MM1.R, OPM1 , and U266 myeloma cells. B) Effect of cyproheptadine (20 μM) on cyclin D1 , D2, and D3 in AML2, AML3, HL60, OCI-M2, and NB4 leukemia cells. C) Effect of increasing concentrations of cyproheptadine on cyclin D2 in My5 and KMS11 myeloma cells.

Figure 6 is a series of graphs demonstrating that cyproheptadine arrests cell lines in the G1 phase of the cell cycle. A) LP1 myeloma cells twenty four (black) and forty-eight (gray) hours after cyproheptadine treatment, the percentage of cells in each phase of the cell cycle was determined by Pl staining and flow cytometry. B) KMS11 , My5, and LP1 MM cell lines twenty four hours after cyproheptadine treatment, the percentage of cells in each phase of the cell cycle was determined by Pl staining and flow cytometry. C) AML2, CEM, and Jurkat leukemia cell lines twenty four hours after cyproheptadine treatment, the percentage of cells in each phase of the cell cycle was determined by Pl staining and flow cytometry.

Figure 7 is a series of graphs demonstrating that cyproheptadine reduces the viability of myeloma and AML cell lines. A) Cell viability of myeloma cell lines 72 hrs after cyproheptadine treatment by MTS assay. B) Cell viability of AML cell lines 72 hrs after cyproheptadine treatment by MTS assay. C) Cell viability of AML2 cell lines 24, 48, and 72 hours after cyproheptadine treatment by MTS assay. D) Cell viability of LP1 cell lines 24, 48, and 72 hours after cyproheptadine treatment by MTS assay. E) Cell viability of primary myeloma (MM), acute myeloid leukemia (AML), and normal hematopoietic stem cells (N-PBSC) treated for 48 hours. After treatment, apoptosis was measured by staining with Annexin V-FITC. Mononuclear cells from the marrow of patients with multiple myeloma were co-stained with PE- labeled anti-CD138 to identify the plasma cells and the percentage of CD138⁺/Annexin V^~ cells was quantified as a marker of myeloma cell viability. F) Colony formation of primary normal hematopoietic stem cells and AML cells after 24 hours of treatment with increasing concentrations of cyproheptadine

Figure 8 is a series of graphs that shows treatment with cyproheptadine abolishes the formation of malignant ascites in mouse.

Figure 9 is a graph that shows treatment with cyproheptadine decreases growth of myeloma tumors in mice. Figure 10 is a series of graphs and immunoblots that shows treatment with cyproheptadine activates the mitochondrial pathway of caspase activation. A) Pretreatment with the pan-caspase inhibitor z-VAD-fmk (100 μM) prevents cyproheptadine-induced cell death by an MTS assay 48 hours after treatment. B) Activation of caspases 3, 8 and 9 by immu nob lotting after treatment of cell lines MY5, OPM1 , AML2, and CEM with increasing concentrations of cyproheptadine (CYP). C) Changes in mitochondrial membrane potential (ΔψM) and Pl staining in U266 and NB4 cell lines at increasing times after treatment with cyproheptadine (20 μM). D) Changes in mitochondrial membrane potential (ΔψM) and Pl staining in MDAY-D2 and LP1 cell lines 48 hours after treatment with increasing concentrations of cyproheptadine.

Figure 11 is a series of graphs that shows treatment with cyproheptadine induces cell death through a mechanism independent of competitive inhibition of the histamine and serotonin receptors. AML1 , CEM, OCI MY5, KSM11 , AML2, and OPM1 cell lines were pre-treated with 10 μM of histamine, serotonin, or histamine and serotonin, or buffer for 24 hours. After pretreatment, cells were incubated with increasing concentrations of cyproheptadine for an additional 48 hours. After incubation, cell viability was measured by MTS assay.

Figure 12 is a series of graphs that shows treatment with the structural cyproheptadine-analogs and tripolidine. A) The chemical structures of cyproheptadine, cyclobenzaprine, amitriptyline, loratadine, and triprolidine. B) KMS11 myeloma cells, LP1 myeloma cells, Jurkat leukemia cells, and OCI- AML2 leukemia cells were treated with increasing concentrations of cyproheptadine (CYP), cyclobenzaprine (CBZ), amitriptyline (AMIT), loratadine (LOR), and triprolidine (TPL). Seventy-two hours after incubation, cell viability was measured by MTS. C)

Figure 13 is a series of immunoblots that shows KSM11 and LP1 myeloma cells when treated with increasing concentrations of cyproheptadine. After treatment, cells were harvested, lysed, and levels of SP-1 , AP2A, CEBPA, Cyclin D2 (CCND2) and beta-actin were measured by immunoblotting.

DETAILED DESCRIPTION OF THE APPLICATION

It has been demonstrated that cyproheptadine inhibits D-cyclin expression and induces cell death in proliferative diseases involving increased expression of D-cyclins. It has also been shown that cyproheptadine and its analogs induce cell death in hematological malignant cells, including a wide variety of cell lines and patient cancer cells. Further it has been demonstrated that cyproheptadine abolishes formation of malignant ascites in an in vivo mouse model and decreases tumor volume of established tumors in mice. The mechanism of action of the compounds described herein has been demonstrated to not involve competitive inhibition of histamine and serotonin receptor pathways, but rather an alternate pathway. It has further been demonstrated that the doses required to achieve a therapeutic effect are in the low micromolar range, for example in the range of about 5 micromolar to about 50 micromolar, or about 10 micromolar to about 20 micromolar. Doses comparable to this range are attainable in vivo. Accordingly, the compounds and pharmaceutical compositions presented herein are useful for treating proliferative diseases involving increased expression of D-cyclins and/or hematological malignancies in patients. I. Definitions

The term "Chalky!" as used herein means straight and/or branched chain, saturated alkyl radicals containing from one to four carbon atoms and includes methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl and t-butyl.

The term "fluoro-substituted" with respect to any specified radical as used herein means that the one or all of the hydrogen atoms in the radical have been replaced with a fluorine, and includes trifluoromethyl, pentafluoroethyl, fluoromethyl and the like.

The term

as used herein means straight and/or branched chain, saturated alkoxy radicals containing from one to four carbon atoms and includes methoxy, ethoxy, propoxy, isopropoxy, and the like.

The term "pharmaceutically acceptable" means compatible with the treatment of animals, in particular, humans.

The term "pharmaceutically acceptable salt" means an acid addition salt which is suitable for or compatible with the treatment of patients.

The term "pharmaceutically acceptable acid addition salt" as used herein means any non-toxic organic or inorganic salt of any base compound of the application. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p- toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. The term "solvate" as used herein means a compound of the application, or a pharmaceutically acceptable salt thereof, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate".

The present application includes within its scope, methods and uses including prodrugs of the compounds of the application. In general, such prodrugs will be functional derivatives of the compound which are readily convertible in vivo into the compound from which it is notionally derived. Prodrugs may be conventional esters formed with available hydroxy, or amino group. For example, an available OH or NH group in a compound may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C₈-C^) esters, acyloxymethyl esters, carbamates and amino acid esters. In further embodiments, the prodrugs are those in which one or more of the hydroxy groups in the compounds is masked as groups which can be converted to hydroxy groups in vivo. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985.

The term "cyproheptadine" as used herein means 4-(5H- dibenzo[a,d]cyclohepten-5-ylidene)-1-methylpiperidine and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The term "amitriptyline" as used herein means 3-(10,11-dihydro-5H- dibenzo[[a,d]]cycloheptene-5-ylidene)-N,N-dimethyl-1-propanamine and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The term "loratadine" as used herein means ethyl-4-(8-chloro-5,6- dihydro-11 H-benzo[5,6]cyclohepta[1 ,2-b]pyridin-11 -ylidine)-1- piperidinecarboxylate and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The term "cyclobenzaprine" as used herein means 3-(5H- dibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-1-propanamine and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The term "compound(s) of the application" as used herein means compound(s) of Formula I, and/or pharmaceutically acceptable salts, solvates and/or prodrugs thereof. It should be noted that the methods and uses extend to cover mixtures of compounds of Formula I and their pharmaceutically acceptable salts, solvates and/or prodrugs.

The term "treating" or "treatment" as used herein means administering to a subject a therapeutically effective amount of the compound of the present application and may consist of a single administration, or alternatively comprise a series of applications. For example, the compound of the present application may be administered at least once a week. However, in another embodiment, the compound may be administered to the subject from about one time per week to about once daily for a given treatment. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration and the activity of the compounds of the present application, or a combination thereof. In one embodiment, the treatment is chronic treatment and the length of treatment is 1-2 weeks, 2-4 weeks or more than 4 weeks. The treatment regimen can include repeated treatment schedules. It will also be appreciated that the effective amount or dosage of the compound used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. As used herein, and as well understood in the art, "treatment" or

"treating" is also an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment also includes palliative treatment. Further any of the treatment methods or uses described herein can be formulated alone or for contemporaneous administration with other agents that treat proliferative disorders involving increased cyclin D expression, and/or hematological malignancies including leukemia and multiple myeloma.

The term "chronic treatment" as used herein refers to treatment that is administered repeatedly and lasts at least 1 week, 1-2 weeks, 2-4 weeks or more than 4 weeks, including indefinitely, or for the rest of the subject's life or until a desired treatment outcome is reached. Administration is optionally daily, twice daily or once about every two days, or more days.

The term "palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.

The term "prevention" or "prophylaxis", or synonym thereto, as used herein refers to a reduction in the risk or probability of a patient becoming afflicted with cancer or manifesting a symptom associated with cancer. For example a subject diagnosed with a precancerous proliferation may be administered a compound of the application to prevent the occurrence of cancer. For example, a patient diagnosed with MGUS, a precancerous clonal expansion of plasma cells, may be administered a compound of the application to prevent or delay the progression to multiple myeloma.

As used herein, the phrase "effective amount" or "therapeutically effective amount" or a "sufficient amount" of a compound or composition of the present application is a quantity sufficient to, when administered to the subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount" or synonym thereto depends upon the context in which it is being applied. For example, in the context of treating a hematologicial malignancy or a proliferative disease involving increased expression of D-cyclins, it is an amount of the compound sufficient to achieve a treatment response as compared to the response obtained without administration of the compound. The amount of a given compound of the present application that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g. age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art. Also, as used herein, a "therapeutically effective amount" of a compound of the present disclosure is an amount which results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a compound of the present disclosure may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regime may be adjusted to provide the optimum therapeutic response.

The term "subject" as used herein includes all members of the animal kingdom including mammals, suitably humans.

The term "proliferative disease involving increased expression of D- cyclins" means any disease wherein a cell type increases in numbers and has increased expression of cyclin D1 , D2, and or D3. One skilled in the art would readily understand that D-cyclin expression is easily detected by methodologies known in the art such as protein detection methods such as immunoblotting and ELISA and nucleic acid methods such as RT-PCR and northern analysis. Increased D-cyclin expression can be determined by comparing the level of D-cyclin expression to one or more control samples, individually or pooled. The term "hematological malignancy" as used herein means a cancer of the blood and includes without limitation all types of cancer that affect blood, bone marrow and lymph node. In one embodiment the hematological malignancy is a non-solid tumor. Hematological malignancies include leukemias, lymphomas and multiple myeloma. Hematological malignancies as used herein optionally have or do not have increased D-cyclin expression.

The term "leukemia" as used herein means any disease involving the progressive proliferation of abnormal leukocytes found in hematopoietic tissues, other organs and usually in the blood in increased numbers.

Leukemia includes acute myeloid leukemia also referred to as acute myelogenous leukemia, and acute lymphocytic leukemia.

The term "AML", "acute myeloid leukemia" or "acute myelogenous leukemia" as used herein, refers to a cancer of the myeloid line of white blood cells and includes promyelocytic leukemia (APL) as well as the French- American-British (FAB) classification system subtypes, MO-M7, and the WHO categories and subcategories.

The term "ALL" or "acute lymphocytic leukemia" as used herein, refers to a cancer of the lymphocyte lines of white blood cells and includes the FAB classification subtypes and WHO categories and subcategories.

The term "multiple myeloma" as used herein means any tumor or cancer composed of cells derived from the hematopoietic tissues of the bone marrow that secrete antibody such as plasma cells. Multiple myeloma is also referred to as MM, myeloma, plasma cell myeloma, or as Kahler's disease and includes symptomatic myeloma, asymptomatic myeloma and MGUS (monoclonal gammopathy of undetermined significance).

The term "a cell" as used herein includes a plurality of cells. Administering a compound to a cell includes in vivo, ex vivo and in vitro treatment.

As used herein, to "inhibit" or "suppress" or "reduce" expression or activity, such as cyclin D expression, is to reduce the expression or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. The terms "inhibitor" and "inhibition", in the context of the present application, are intended to have a broad meaning and encompass a compound of Formula I, and/or a pharmaceutically acceptable salt, hydrate and/or prodrug thereof, which directly or indirectly (e.g., via reactive intermediates, metabolites and the like) acts decreases cyclin D expression.

The term "cell death" as used herein includes all forms of cell death including necrosis and apoptosis. In understanding the scope of the present disclosure, the term

"comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

The term "solid dosage form" is to be understood to refer to individually coated tablets, capsules, granules or other non-liquid dosage forms suitable for oral administration. It is to be understood that the solid dosage form includes, but is not limited to, non-controlled release, controlled release and time-controlled release dosage form units, employed preferably in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet.

The term "liquid dosage form" is to be understood to refer to non-solid dosage forms suitable for, but not limited to, intravenous, subcutaneous, intramuscular, or intraperitoneal administration. Solutions of a compound of the application can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

II. Method/Uses for Treating Proliferative Disorders and Hematological malignancies D-Cyclins are over-expressed in virtually all cases of multiple myeloma

(MM) and contributes to its pathogenesis and chemoresistance (1-3). In addition, D-cyclins are increased in a subset of high-risk patients with acute myleoid leukemia (AML) (1). Pharmacological and/or genetic inhibition of D- cyclins may induce apoptosis in MM and AML cells and serve as a basis for novel therapeutics.

To this end, novel therapeutics for treating proliferative diseases involving increased expression of D-cyclins and hematological malignancies such as MM, ALL and AML have been identified. Using a chemical biology screen for inhibitors of cyclin D2 transactivation, the appetite stimulant cyproheptadine has surprisingly been identified as an inhibitor of cyclin D2 transactivation. It was demonstrated that cyproheptadine inhibited D-cyclin transactivation and induced apoptosis in myeloma and leukemia cell lines and patient sample cells, as well as in established in vivo mouse models. Pretreatment of cells with histamine or serotonin did not diminish the apoptotic effect of cyproheptadine demonstrating that cyproheptadine induced therapeutic effects are independent of histamine and serotonin pathways. Further, the structurally related compounds amitriptyline, cyclobenzaprine, and loratadine were shown to induce cell death in leukemic cells lines.

The present application therefore includes a method for treating proliferative diseases involving increased expression of D-cyclins and/or hematological malignancies comprising administering to a subject in need thereof, a compound selected from a compound of Formula I, and salts, solvates and prodrugs thereof:

wherein

.-TTTT^ is a single or double bond,

R¹ is selected from H, C^alkyl, C(O)OCi-₄alkyl, C(O)Ci-₄alkyl and C(O)NHCi-

₄alkyl; R² is selected from H and

R³ is selected from H and C-_Malkyl; or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring;

R⁴ and R⁵ are independently selected from H, halogen, hydroxy, C^alkyl, fluoro-substituted C-i^alkyl and Ci-4alkoxy; and

X is selected from C and N.

The present invention further includes a use of a compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof, for treating proliferative diseases involving increased expression of D-cyclins and/or hematological malignancies as well as a use of a compound selected from a compound of Formula I as defined above, and salts, solvates and prodrugs thereof, to prepare a medicament for treating proliferative diseases involving increased expression of D-cyclins and/or hematological malignancies. In one embodiment of application the methods and uses are directed to the treatment of a proliferative disease involving increased D-cyclin expression. In another embodiment of application the methods and uses are directed to the treatment a hematological malignancy. In one embodiment the hematological malignancy is a leukemia such as acute myeloid leukemia or acute lymphocytic leukemia. In another embodiment the hematological malignancy is multiple myeloma.

In the methods and uses of the present application, the compounds of Formula I include those in which R¹ is selected from H, Ci.₄alkyl, C(O)OCi- ₄alkyl, C(O)Ci-₄alkyl and C(O)NHCi-₄alkyl. In an embodiment of the application, R¹ is selected from H, Ci^alkyl and C(O)OCi_-4alkyl, suitably R¹ is selected from CH₃ and C(O)OC-|.₂alkyl. Further, the compounds of Formula 1 include those in which R² and R³ are independently selected from H and Ci- ₄alkyl or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring. In an embodiment of the application R² and R³ are independently selected from H and CH₃, suitably CH₃, or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring. Still further, the compounds of Formula I include those in which R⁴ and R⁵ are independently selected from H, halogen, hydroxy, Ci^alkyl, fluoro-substituted Ci^alkyl and Ci^alkoxy. In an embodiment of the present application and R⁴ and R⁵ are independently selected from H, Cl, F, I, hydroxy, CH₃, CF₃, and and CH₃O.

The compounds of the application may have at least one asymmetric centre. Where the compounds according to the application possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be understood that while the stereochemistry of the compounds of the application may be as provided for in any given compound listed herein, such compounds of the application may also contain certain amounts (e.g. less than 20%, preferably less than 10%, more preferably less than 5%) of compounds of the application having alternate stereochemistry. Compounds of Formula I₁ such as cyproheptadine, amitriptyline, cyclobenzaprine, and loratadine are available commercially. Compounds of the application are also prepared using methods known in the art, for example, as described in United States patent number 3,014,911 "Derivatives of Dibenzo[a,c]cyclo-heptatriene".

The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method. The formation of solvates of the compounds will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. In an embodiment of the application, the compound of Formula I is selected from cyproheptadine, amitriptyline, cyclobenzaprine and loratadine, and pharmaceutically acceptable salts, solvates and/or prodrugs thereof. In a further embodiment of the application, the compound of Formula I is cyproheptadine and/or a pharmaceutically acceptable salt, solvate and/or prodrug thereof. In an embodiment of the application, cyproheptadine is cyproheptadine hydrochloride.

In another embodiment, the compound of Formula I is amitriptyline and/or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In another embodiment, the compound of Formula I is cyclobenzaprine and/or a pharmaceutically acceptable salt, solvate or prodrug thereof.

In another embodiment, the compound of Formula I is loratadine and/or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Further, the inventors have also demonstrated that cyproheptadine inhibits D-cyclin expression. Accordingly, the application describes a method of inhibiting D-cyclin expression in a cell or in a subject, comprising administering to the cell or subject, a compound selected from a compound of Formula I as defined above and pharmaceutically acceptable salts, solvates and prodrugs thereof. Inhibiting D-cyclin expression means in one embodiment, reducing expression of at least one D-cyclin, including cyclin D1 , cyclin D2 or cyclin D3, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% as determined using assays known in the art, for example by northern analysis or immunoblotting. It has been demonstrated that the compounds of Formula I induce cell death in leukemia and myeloma cells. Accordingly, the application also includes a method of inducing cell death in a leukemia cell or a myeloma cell comprising administering to the cell, a compound of Formula I as defined above and/or a pharmaceutically acceptable salt, hydrate and/or prodrug thereof.

III. Compositions A further aspect of the application is a pharmaceutical composition for the treatment of proliferative diseases involving increased expression of D- cyclins and/or hematological malignancies comprising a compound selected from a compound of Formula I and a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier in a dosage form, wherein the compound of Formula I is

wherein

^= is a single or double bond,

R¹ is selected from H, C_1-4alkyl, C(O)OC_1-4alkyl, C(O)Ci-4alkyl and C(O)NHC_1-

₄alkyl; R² is selected from H and Ci^alkyl;

R³ is selected from H and C-ualkyl; or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring;

R⁴ and R⁵ are independently selected from H, halogen, hydroxy, C-Malkyl, fluoro-substituted Ci^alkyl and C-_Malkoxy; and

X is selected from C and N.

In a further embodiment, the pharmaceutical composition is formulated for oral administration or injection.

In the compositions and medicaments described herein, the compounds of Formula I include those in which R¹ is selected from H, Ci-

₄alkyl, C^Od^alkyl, C(O)Ci-₄alkyl and C(O)NHCi-₄alkyl. In an embodiment of the application, R¹ is selected from H, Ci^alkyl and C(O)OCi-₄alkyl, suitably

R¹ is selected from CH₃ and C(O)OCi-₂alkyl. Further, the compounds of Formula 1 include those in which R² and R³ are independently selected from H and C-ualkyl or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring. In an embodiment of the application R² and R³ are independently selected from H and CH₃, suitably CH₃, or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring. Still further, the compounds of Formula I include those in which R⁴ and R⁵ are independently selected from H, halogen, hydroxy, C-ualkyl, fluoro-substituted Ci-4alkyl and

In an embodiment of the present application and R⁴ and R⁵ are independently selected from H, Cl₁ F, I, hydroxy, CH₃, CF₃, and CH₃O.

In an embodiment of the application, the compound of Formula I are selected from cyproheptadine, amitriptyline, cyclobenzaprine and loratadine, and pharmaceutically acceptable salts, solvates and/or prodrugs thereof. In a further embodiment of the application, the compound of Formula I is cyproheptadine and/or a pharmaceutically acceptable salt, solvate and/or prodrug thereof.

The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.

In an embodiment of the disclosure the pharmaceutical composition contains about 0.01% to about 1%, suitably about 0.01% to about 0.5%, of one or more compounds of the application. The composition may be prepared, for example, by mixing the carrier and the compound(s) at a temperature of about 40 ⁰C to about 70 ⁰C, the composition retains stability in solution for 3 years at temperatures up to about 25 ⁰C.

Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences. On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3- dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesyl- phosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient. The compositions of the application can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration. For parenteral administration, solutions of a compound described herein can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations.

Wherein the route of administration is oral, the dosage form may be for example the composition may be incorporated with excipient and used in the form of enteric coated tablets, caplets, gelcaps, capsules, ingestible tablets, buccal tablets, troches, elixirs, suspensions, syrups, wafers, and the like.

A further aspect of the application is a composition formulated for as an oral dosage form selected from enteric coated tablets, caplets, gelcaps, and capsules, comprising from about 5 to less than about 2000 mg, suitably from about 10 to about 100 mg, about 30 to about 60 mg, or about 35 to about 50 mg, of a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate or prodrug thereof and a pharmaceutically acceptable carrier. Suitably each tablet, caplet, gelcap or capsule comprises about 2 to about 2000 mg, suitably about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg or about 35 to about 50 mg of the compound.

In an embodiment, the dosage form is solid or liquid.

A further aspect of the application is a pharmaceutical composition wherein a solid dosage form contains from about 2 to about 2000 mg of a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate and prodrug thereof, suitably from about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg, about 30 to about 60 mg or about 35 mg to about 50 mg the compound. A further aspect of the application is a pharmaceutical composition wherein the dosage form is a liquid dosage form that contains from about 2 to about 2000 mg a compound selected from a compound of Formula I as defined above and a pharmaceutically acceptable salt, solvate or prodrug thereof, suitably from about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg, about 35 to about 50 mg, of the compound. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerin.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

The dosage form comprises an effective amount or a therapeutically effective amount. In one embodiment the dosage form comprises about 5 to about 2000 mg of a compound of Formula I as defined above, and/or pharmaceutically acceptable salt, solvate and/or prodrug thereof. In another embodiment, the dosage form comprises about 10 to about 100 mg of the compound.

A further aspect of the application is a pharmaceutical composition comprising a compound selected from a compound of Formula I as defined above, and a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier in unit dosage form in an amount suitable to provide about 0.1 to about 200 mg /kg body weight, suitably about

0.1 to about 100 mg/kg body weight, about 0.1 to about 80 mg/kg body weight, about 0.1 to about 40 mg/kg/body weight, about 0.1 to about 20 mg/kg body weight about 0.1 to about 2mg/kg body weight or about 0.5 to about 2 mg /kg body weight, formulated into a solid oral dosage form, a liquid dosage form, or an injectable dosage form.

A further aspect of the application is a composition, wherein the amount of the compound of Formula I, and/or pharmaceutically acceptable salt, solvate and/or prodrug thereof, is an effective amount for treatment of acute myeloid leukemia, acute lymphocytic leukemia or multiple myeloma.

The pharmaceutical compositions are useful for treating proliferative diseases involving increased expression of D-cyclins, hematological malignancies including AML, ALL and MM. Accordingly, also included are methods of treating a proliferative disease involving increased expression of a

D-cyclin comprising administering an affective amount of one of the pharmaceutical compositions of the application to a subject or cell. A further aspect of the application is a pharmaceutical composition for treatment of hematological malignancies, including acute myeloid leukemia, acute lymphocytic leukemia or multiple myeloma, in a subject, which composition comprises as active ingredient a compound of Formula I as defined above, and/or a pharmaceutically acceptable salt, solvate or prodrug thereof, a pharmaceutically acceptable carrier in unit dosage form, wherein the pharmaceutical composition is suitable for oral administration or injection.

Compounds of Formula I, and/or pharmaceutically acceptable salt, solvate and/or prodrug thereof, are also useful for the preparation of a medicament for the treatment of a proliferative disorder involving increased D- cyclin expression, or for the treatment of a hematological malignancy such as leukemia including AML and ALL, or MM.

Accordingly an additional aspect disclosed herein, is the use of a compound of Formula I as defined above, and/or a pharmaceutically acceptable salt, solvate and/or prodrug thereof, in the preparation of a medicament for treating a proliferative disorder involving increased D-cyclin expression, and/or for the treatment of a hematological malignancy. In certain embodiments the hematological malignancy is leukemia. In one embodiment the leukemia is acute myeloid leukaemia. In another embodiment, the leukaemia is acute lymphocytic leukaemia. In a further embodiment the haematological malignancy is multiple myeloma. In certain embodiments, the compound of Formula I, and/or pharmaceutically acceptable salt, solvate and/or prodrug thereof, is used to manufacture a medicament wherein the effective amount of the compound is within the range of about 0.1 to about 200 mg/kg body weight, suitably in the range of about 0.1 to about 100 mg/kg body weight, about 0.1 to about 80 mg/kg body weight, about 0.1 to about 40 kg/ body weight, about 0.1 to about 20 mg/kg, about 0.1 to about 2 mg/kg, or about 0.5 to about 2 mg/kg body weight. A further aspect of the application is a commercial package comprising a composition according to the present application, and associated therewith instructions for the use thereof for treatment of proliferative diseases involving increased expression of D-cyclins or hematological malignancies such as leukemias including acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL), and multiple myeloma (MM).

IV. Methods of Screening for Cyclin D Modulators

The inventors have developed a high throughput chemical genomics screen for identifying modulators of D-cyclins. The method allows identification of c-maf dependent and c-maf independent inhibitors of cyclin D2. Accordingly, in one embodiment, the application provides a method of identifying D-cyclin modulators comprising: i) treating cells over-expressing a c-maf cassette and a cyclin D promoter driving a reporter cassette, with a test compound; ii) treating cells over-expressing a c-maf cassette and a cyclin D promoter driving a reporter cassette without the test compound; iii) comparing reporter cassette activity and cell viability in cells receiving the test compound to cells not receiving the test compound wherein a Iog2 ratio of (reporter activity in cells receiving the test compound/reporter activity in cells not receiving the test compound)/viability of cells receiving the test compound/viability of cells not receiving the test compound) that is less than -1 is indicative of the test compound being an inhibiting modulator and wherein a ratio of greater than 1 is indicative of the test compound being an activating modulator. The cyclin D promoter in one embodiment comprises -877 bp to -4 bp of the cyclin D2 promoter. In one embodiment the method of identifying cyclin D modulators comprises the method illustrated in Figure 1. In another embodiment the method comprises the method illustrated in Figure 3.

The following non-limiting examples are illustrative of the present application: EXAMPLES

Materials and Methods

Cell culture, constructs and transduction

Mouse fibroblast NIH3T3 cells were maintained in Dulbeco's Modified Eagle's medium plus 10% calf serum (Hyclone, Logan, Utah). Myeloma cell lines and leukemia cell lines were grown in Iscove's modified essential medium (IMEM) plus 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). All the media were supplemented with 1mM glutamate and antibiotics. Cells were cultured at 37⁰C with 5% CO2 in a humid incubator. Primary human MM and acute myeloid leukemia (AML) samples were isolated from fresh bone marrow and peripheral blood samples, respectively, and obtained from patients who consented to the donation of a research sample. Primary normal hematopoietic cells were obtained from healthy volunteers donating their peripheral blood mononuclear stem cells (PBSC) for allotransplantation. Mononuclear cells were isolated from the samples by Ficoll density centrifugation. Primary cells were cultured at 37°C in IMDM supplemented with 10% FCS, 1 mM L-glutamine and appropriate antibiotics.

Full-length c-maf cDNA was subcloned into an IRES-GFP-MIEV retroviral vector. NIH3T3 cells were infected with this construct and stable cells expressing GFP and c-maf were selected by flow cytometry and immunoblotting, respectively. The full-length c-maf was also subcloned into a pcDNA3.1 vector under the control of a CMV promoter.

The promoter of cyclin D2 (-894 to -4), containing c-maf responsive element sequence (MARE), was cloned from HeLa cell genomic DNA and subcloned into the pGL2 luciferase reporter vector (Promega, Madison, Wl). This construct was co-transfected with pcDNA3.1 containing a neomycin resistance gene into NIH3T3 wild type cells and NIH3T3 cells stably over- expressing c-maf-IRES-GFP. Cells stably expressing c-maf, GFP, and luciferase were selected for further application. High throughput screen for inhibitors of cyclin D2 transactivation

NIH3T3 cells stably expressing c-maf and the cyclin D2 promoter driving luciferase (13,000 cells per well) were plated in 96-well plates by the Biomek FX liquid handler (Beckman, Fullerton, CA). The same workstation was used for plate formatting and reagent distribution. After the cells had adhered (6hr after plating), they were treated with aliquots of molecules from LOPAC (Sigma, St. Louis, MO) and Prestwick (Prestwick Chemical Inc, lllkirch, France) libraries. Final concentration of LOPAC compounds was 5 μM (0.05% DMSO) while for the Prestwick library, 10ng of each sample was added, resulting in an average final concentration of approximately 5 μM (0.1% DMSO). Control wells, treated with vehicle alone containing consistent levels of DMSO, were distributed in the first and last columns of the plate to monitor signal variability. Cells were incubated with the molecules at 37⁰C for 20 hours. After incubation, cyclin D2 transactivation was assessed by the luciferase assay and viability was assessed by the MTS assay. Luciferase assay

Luciferase activity was assessed according to the manufacturer's instructions (Promega, Madison, Wl). Briefly, the cell culture medium was removed using an EMBLA plate washer (Molecular Devices, Sunnyvale, CA) and 1X GIo Lysis buffer (Promega) was added by the robotic liquid handler. After 10 min incubation, an equal volume of Bright-Glo Luciferase substrate (Promega) was added and the luminescence signal was detected with a 96- well Luminoskan luminescence plate reader (Thermo Labsystem, Waltham, MA) with a 5 second integration. Ce// Viability assays

Cell viability was assessed by the MTS assay (Promega) according to manufacturer's instructions and as previously described (2). In primary cells, cell viability and apoptosis was measured by Annexin

V-FITC (Biovision Research Products, Mountain View, CA) staining and flow cytometry according to manufacturer's instructions and as previously described (3). Mononuclear cells from patients with multiple myeloma were co-stained with PE-labeled anti-CD138 and FITC-labeled Annexin V. The percentage of CD138⁺/Annexin V^" cells was quantified as a marker of cell viability as previously described (4). To assess clonogenic growth, primary AML cells or G-CSF mobilized peripheral blood stem cells (PBSC) (6.25x1 O^s/ml) were treated with test compound or buffer control for 24 hours. After treatment, cells were washed and equal volumes were plated in triplicate in MethoCult GF H4434 medium (StemCell Technologies Inc., Vancouver, BC, Canada) containing 1 % methycellulose in IMDM, 30% FCS, 1% Bovine Serum Albumin, 3 U/ml recombinant human Erythropoietin, 10^"4M 2-Mercaptoethanol, 2 mM L- glutamine, 50 ng/ml recombinant human stem cell factor, 10 ng/ml GM-CSF, and 10 ng/ml rh IL-3. Seven days (AML samples) or 14 days (normal PBCS) after plating, the number of colonies containing 20 or more cells was counted as described previously. lmmunoblotting

Whole cell lysates were prepared from myeloma and leukemia cells as described previously (2). Briefly, cells were washed with phosphate-buffered saline (PBS, pH 7.4) and re-suspended in lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCI, 0.1% Triton X-100, 0.5% sodium deoxycholate, and 5 mM EDTA] containing protease inhibitors (Complete tablets, Roche, Indianapolis, IN). Protein concentrations were determined by the Bradford assay. Equal amounts of protein were subjected to SDS-PAGE gels followed by transfer to PVDF membranes. Membranes were probed with antibodies including monoclonal anti-human cyclin D1 (1 :200 v/v) (Cell Signaling Technology, Inc), polyclonal anti-human cyclin D2 (1 :400 v/v) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti-human cyclin D3 (1 :200 v/v) (BD Pharmingen, San Jose, CA), polyclonal anti-human caspase-3 (1 :5,000 v/v) (Cell Signaling Technology, Inc. Danvers, MA), monoclonal anti-human caspase-9 (1 :3,000 v/v) (R&D Systems, Minneapolis, MN), monoclonal anti- human caspase-8 (1:3,000 v/v) (Pharmingen, San Jose, CA), or monoclonal anti-beta actin (1 :10,000 v/v) (Sigma) followed by secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse (1 :10,000 v/v) or anti-rabbit IgG (1: 5,000 v/v) (Amersham Bioscience UK, Little Chalfont, England). Detection was performed by the Enhanced Chemical Luminescence (ECL) method (Pierce, Rockford, IL). CeII cycle analysis

Cell cycle analysis was performed as previously described (5). Briefly, cells were harvested, washed with cold PBS, re-suspended in 70% cold ethanol and incubated overnight at -20 ⁰C. Cells were then treated with 100 ng/ml of DNase-free RNase (Invitrogen, Carlsbad, CA) at 37 ⁰C for 30 min, washed with cold PBS, and resuspended in PBS with 50 μg/ml of propidium iodine (Pl). DNA content was analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Florida, USA). The percentage of cells in each phase of the cell cycle was calculated with ModFit software (Verity Software House, Topsham, ME).

Mitochondrial membrane potential (ΔΨM) assessment

Changes in mitochondrial membrane potential (ΔΨ_M) were detected by staining cells with 40 nM of DilCi(5)

(1 ,1'3,3,3',3'hexamethylindodicarbocyanine) (Invitrogen) and 5 μg/ml of propidium iodine as previously described (6). Cells stained with DilCi(5) were incubated at 37 ⁰C for 30 minutes and then analyzed by flow cytometry (Coulter Epics Elite, Beckman-Coulter, Florida) by exciting at 633 nm and measuring through a 675 + 20 nm bandpass filter. Assessment of cyproheptadine's anti-leukemic and anti-myeloma activity in vivo

MDAY-D2 (MDAY) murine leukemia cells (5 X 10⁵) were injected intraperitoneal^ into DBA2 mice (Jackson Laboratory, Bar Harbor, Maine). Mice were then treated with cyproheptadine at 50 mg/kg/day in PBS with 2% ethanol or vehicle control intraperitoneal^ for 5 days. Ten days after injection of cells, mice were sacrificed, and the volume and cell count in the malignant ascites was measured.

LP1 multiple myeloma cells (10X10⁶) were injected s.c. into the flank of sublethally irradiated (3.5 Gy) NOD/SCID mice (Ontario Cancer Institute, Toronto, Canada). When tumors where palpable, mice were treated with 36 mg/kg of cyproheptadine daily in PBS with 2% DMSO or vehicle control intraperitoneally daily for 7 weeks. Tumor volume (tumor length x width² x 0.5236) (7) was measured weekly using calipers. Mouse body weight and blood counts were also monitored weekly.

A high throughput screen identifies c-maf dependent and independent inhibitors of the cyclin D2 promoter One of the regulators of cyclin D2 is the oncogene c-maf that is also frequently over-expressed in MM (8). Therefore, the inventors sought to identify c-maf dependent and independent inhibitors of cyclin D2. To identify such small molecule inhibitors, the inventors developed a high throughput chemical genomics screen (Figures 1 to 3). NIH 3T3 cells stably over- expressing a c-maf-IRES-GFP cassette in an MIEV vector and the cyclin D2 promoter (-877 bp to -4 bp) driving firefly luciferase were seeded in 96 well plates by a robotic liquid handler. After the cells had adhered to the plates they were treated with aliquots of the LOPAC (1280 compounds) and Prestwick (1120 compounds) libraries of drugs and chemicals. Compounds were tested at a final concentration of ~5 μM and <0.01% DMSO. Sixteen hours after the addition of the compounds, luciferase expression was measured as a marker of cyclin D2 transactivation. In parallel, cell viability was measured with an MTS assay. Thus, each compound from the library was tested in two assays - luciferase for cyclin D2 activity and MTS for viability. The results of the screens are shown in Figure 2. The activity of the compound is expressed as log 2((sample luciferase RFU/control luciferase RFU)/(sample MTS OD/control MTS OD)). Compounds with an activity <-1 were considered inhibitors and compounds with an activity >1 were considered activators. The work flow diagram of the assay and the screen is shown in Figure 3. Hits were empirically defined as compounds that preferentially reduced luciferase activity over reductions in viability and defined mathematically as log 2((sample luciferase /control luciferase)/(sample MTS OD/control MTS OD)) <-1 , which corresponds to a 50% reduction in relative luciferase expression. With this definition, the inventors identified 32 reproducible and unique hits (Figure 4A). Using algorithms derived from microarray gene analysis, the inventors determined that the Corticosteroids (CS) family of drugs inhibited the cyclin D2 promoter (Figure 4A). Twenty four of the 26 CS in the libraries were identified as hits in this assay. To identify non-specific inhibitors of luciferase, hits were tested in 3T3 cells over-expressing luciferase driven by an RSV promoter. One compound down regulated luciferase non-specifically and the remaining 31 unique compounds, including cyproheptadine, preferentially inhibited the cyclin D2 promoter (Figure 4A,B). To distinguish between c-maf dependent and independent inhibitors, hits were tested in NIH 3T3 cells expressing the cyclin D2 promoter-Luciferase construct but not c-maf. Seven compounds, including cyproheptadine (Figure 4B), reduced cyclin D2 transactivation equally in cells with or without c-maf expression. These compounds inhibited cyclin D2 transactivation independent of c-maf. Conversely, 24 compounds preferentially decreased cyclin D2 transactivation in the presence of c-maf (Figure 4A).

Specifically determinations of whether molecules identified in the screen were a non-specific inhibitor of luciferase, preferential inhibitors of c-maf-mediated cyclin D2 transactivation, or c-maf independent inhibitors of cyclin D2 transactivation, were made by testing hits (5 μM final) in NIH 3T3 cells over-expressing the RSV promoter driving luciferase, 3T3 cells over- expressing c-maf and the cyclin D2 promoter driving luciferase, and 3T3 cells without c-maf over-expressing the cyclin D2 promoter driving luciferase. Activity of the compounds was expressed as (sample luciferase RFU/control luciferase RFU)/(sample MTS OD/control MTS OD). The numeric value is represented colormetrically in Figure 4A. Drugs were assigned into families based on the annotation from the libraries and clustered using the Cluster and Treeview algorithms.

Corticosteroids inhibit c-maf dependent cyclin D2 transactivation by promoting the proteasomal degradation of c-maf

All of the 24 compounds identified as c-maf-dependant cyclin D2 inhibitors were Corticosteroids (CS) derivatives. The most potent inhibitors were glucocorticoids such as dexamethasone. Mineralocorticoids such as fludrocortisone were weak hits, likely reflecting their weak glucocorticoid activity at higher concentrations. CS inhibited cyclin D2 transactivation in NIH 3T3 cells over-expressing c-maf with an ED₅₀ in the low nanomolar range. In contrast, the ED50 for cyclin D2 transactivation in NIH 3T3 cells with c-maf expression was >10 μM. Subsequently, the inventors demonstrated that CS such as dexamethasone decreased c-maf protein by promoting its ubiquitination and proteasomal degradation by upregulating ubiquitin mRNA through an SP1 -dependent mechanism.

Cyproheptadine inhibits D-cyclin transactivation and arrests celis in the G1 phase

The primary screen also identified the appetite stimulant cyproheptadine as a c-maf independent inhibitor of cyclin D2. Cyproheptadine is an H 1 histamine and serotonin receptor blocker that has been evaluated clinically for the treatment of migraines (9), anorexia (10), and atopic dermatitis (11). In clinical trials, the drug was well tolerated without hematologic toxicity. The ability of cyproheptadine to inhibit D-cyclin expression and induce apoptosis has not previously been reported. To further examine cyproheptadine, the inventors evaluated its effects on D-cyclin expression in myeloma and leukemia cells. Myeloma and leukemia cell lines were treated with cyproheptadine for 24 hours. After incubation, levels of cyclin D1 , D2, and D3 protein were measured by immunoblotting (Figure 5). D-cyclin expression varied among the cell lines, but each cell line expressed at least one D-cyclin. By immunoblotting, cyproheptadine decreased the expression of all of the D-cyclins expressed in the tumor cells. Decreased levels occurred across a range of events that dysregulated D-cyclins including cyclin D1 translocation (U266) (12), c-maf over-expression (OCI-My5) (8, 13) and FGFR3 translocation (KMS11) (14).

Cyproheptadine arrests cells in the G1 phase of the cell cycle

D-cyclins are required for cell commitment to proliferation and entry to the S phase of the cell cycle. Decreasing D-cyclin expression is associated with G1 arrest (15-17). Since cyproheptadine decreased levels of D-cyclins, the inventors tested the effects of cyproheptadine on cell cycle progression. Myeloma and leukemia cells were treated with increasing concentrations of cyproheptadine and the percentage of cells in the different phases of the cell cycle was measured by propidium iodine staining and analysis by flow cytometry. Consistent with its effect on D-cyclin gene and protein expression, cyproheptadine arrested myeloma and leukemia cells in the G0/G1 phase (Figure 6). Cyproheptadine induced cell cycle arrest in a dose dependent manner and at concentrations that were similar to those required to decrease the expression of D-cyclins.

Cyproheptadine induces apoptosis in MM cell lines and primary patient samples

Reductions in D-cyclins and G0/G1 arrest can induce apoptosis in malignant cells (18, 19). Therefore, the inventors tested the effects of cyproheptadine on the viability of myeloma and leukemia cells. Myeloma and leukemia cell lines were treated with increasing concentrations of cyproheptadine and cell viability was measured by the MTS assay after 24, 48, and 72 hours of incubation. Cyproheptadine reduced the viability of myeloma and leukemia cell lines such as LP1 , KMS11 , OCI-AML1 , OCI-AML2 and OCI-AML4 cells at 72 hours with an IC₅O in the low micromolar range. In contrast, it was less toxic to non-leukemic and non-myeloma cells, such as HeLa or NIH3T3 cells with an IC1₀ > 30 μM. Cyproheptadine-induced apoptosis was confirmed by Annexin V-FITC staining. The apoptosis induced by cyproheptadine was concentration- and time-dependent (Figure 7 A to D). The concentration of cyproheptadine required to induce cell death matched the concentrations of cyproheptadine required to decrease D-cyclins and arrest cells in the G0/G1 phase.

Given the effects of cyproheptadine on myeloma and leukemia cell lines, the inventors extended their studies to evaluate the effects of cyproheptadine on primary myeloma and AML samples. Bone marrow and peripheral blood samples were obtained from consenting patients with myeloma and AML, respectively. As controls, normal hematopoietic stem cells were obtained from peripheral blood samples from G-CSF treated volunteers donating peripheral blood stem cells for allotransplant. Mononuclear cells were isolated from these samples and treated with increasing concentrations of cyproheptadine. Forty-eight hours after treatment, cell viability was measured by Annexin V staining. Cyproheptadine reduced the viability of primary myeloma and AML samples, but was less toxic to normal hematopoietic cells (Figure 7E).

In addition to the short-term cytotoxicity assays, the inventors evaluated the effects of cyproheptadine on the clonogenic growth of primary AML and normal hematopoietic cells. Cyproheptadine inhibited the clonogenic survival of the AML samples but not the normal hematopoietic cells (Figure 7F). Cyproheptadine abolishes malignant ascites formation and inhibits tumor growth in xenograft models

Given the selectivity of cyproheptadine cytotoxicity towards malignant cell lines and primary patient samples, the inventors explored its efficacy in vivo using two separate mouse models. DBA2 mice were injected intraperitoneal^ with MDAY-D2 mouse leukemia cells and treated with cyproheptadine (40 mg/kg/day x 5 days) or vehicle control. Significantly, treatment with cyproheptadine completely abolished the formation of malignant ascites in this mouse model (Figure 8 A, B) without decreasing body weight.

In a second experiment, the inventors evaluated the in vivo efficacy of cyproheptadine against a xenograft model of myeloma. LP1 myeloma cells were injected subcutaneously into the flanks of sublethally irradiated NOD/SCI D mice. Two weeks later, once tumors were established, mice were treated with cyproheptadine or vehicle control. In this model, cyproheptadine delayed tumor growth with an approximately two-fold reduction in tumor volume at the end of treatment (Figure 9). Similar results were also obtained in xenograft animals bearing JJN3 myeloma cells. Cyproheptadine did not reduce the animals' body weight or complete blood counts. Cyproheptadine induces apoptosis via the mitochondrial pathway of caspase activation and independent of histamine H1 or Serotonin receptor antagonism

To explore the mechanism of cyproheptadine's toxicity in malignant cells, the inventors first evaluated the effects of the pan caspase inhibitor benzoyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) (Enzyme Systems, Dublin, CA) on cyproheptadine-induced cell death. OCI-MY5 myeloma cells were treated with increasing concentrations of cyproheptadine with and without z-VAD-fmk. Cell viability was measured at 48 hours by MTS assay, z- VAD-fmk completely inhibited cyproheptadine-induced cell death, consistent with the induction of caspase-dependent apoptosis (Figure 10).

To better understand caspase activation by cyproheptadine, the inventors measured levels of pro-caspases 3, 8, and 9 by immunoblotting after treatment with cyproheptadine. In both leukemia and myeloma cell lines, cyproheptadine caused reductions in all three caspases. However reductions in pro-caspases 3 and 9 preceded reductions in pro-caspase-8 (Figures 10B). Activation of caspase-9 is associated with the mitochondrial pathway of caspase activation (20), so the inventors examined the effects of cyproheptadine on the mitochondria. Changes in mitochondrial membrane potential were measured by flow cytometry after treatment with cyproheptadine. In a time- and dose-dependent manner, cyproheptadine caused loss of mitochondrial membrane potential prior to loss of plasma membrane integrity (Figures 10 C, D). These results indicate activation of the mitochondrial caspase pathway by cyproheptadine. Cyproheptadine is a histamine H1 and serotonin 5HT2 receptor antagonist (21 , 22). Therefore, the inventors tested whether the addition of histamine or serotonin in excess could prevent cyproheptadine from inducing apoptosis. Pre-incubation of cells with high concentrations of histamine, serotonin, or a combination of histamine and serotonin did not abrogate cyproheptadine-induced cell death (Figure 11). Therefore, the pro-apoptotic activity of cyproheptadine is not due to competitive inhibition of the H1 and/or serotonin receptors, suggesting that cyproheptadine has an additional molecular target that mediates its cytotoxic function.

To further verify that cyproheptadine does not exert its cell cycle and cytotoxic effects via the histamine or serotonin receptors, the inventors compared its activity with a known anti-histamine, triprolidine. Unlike cyproheptadine, triprolidine did not reduce the viability of leukemia or myeloma cells (Figure 12). Therefore, cyproheptadine's pro-apoptotic effects occur through a mechanism distinct from H1 receptor antagonism.

The cytoxicity of compounds structurally related to cyproheptadine was also tested. KMS11 myeloma cells, LP1 myeloma cells, Jurkat leukemia cells, and OCI-AML2 leukemia cells were treated with increasing concentrations of cyproheptadine, cyclobenzaprine, amitriptyline, loratadine, and triprolidine. After incubation, cell viability was measured by the MTS assay (Figure 12B) Cyproheptadine, cyclobenzaprine, amitriptyline, and loratadine induced cell death in the tested cell lines, but cyproheptadine was the most potent of the tested compounds. In contrast, triprolidine did not induce cell death. Thus, these results demonstrate that compounds of Formula I display the cytotoxic effects of cyproheptadine.

To examine the mechanism by which cyproheptadine decreased levels of D-cyclins, the inventors measured the expression of transcription factors that had binding motifs within the fragment of the cyclin D2 promoter used in the high throughput screen. Treatment of KMS11 and LP1 myeloma cells with cyproheptadine decreased expression of the transcription factor AP2A at times and concentrations associated with reductions in D-cyclin levels (Figure 13). In contrast, levels of CEBPA did not change. Some reduction of SP-1 levels were observed in KMS11 but not LP1 cells. However, the reduction in SP-1 levels in KMS11 cells occurred at concentrations higher than those required to decrease D-cyclin expression. Discussion

Acute myeloid leukemia (AML) and multiple myeloma (MM) are malignant hematological diseases resulting in the proliferation of abnormal cells of myeloid and lymphoid origin, respectively. Both diseases are characterized by poor responses to standard therapies. It would be advantageous for these patients and those with relapsed refractory disease if novel therapies were available. As many of these patients are frail, therapies that achieve an anti-myeloma or leukemia effect without significant toxicity are highly desirable. In malignant cells, multiple studies (including (13-15)) have demonstrated that decreasing D-cyclins arrests cells in the Gi phase of the cell cycle and induces apoptosis. Therefore, the inventors postulated that molecules that reduce D-cyclin expression directly or indirectly could be useful therapeutically for the treatment of hematological malignancies such as multiple myeloma (MM) and acute myeloid leukemia (AML). Such inhibitors might not be toxic to normal cells as normal cells express less cyclin D2 and cycle less rapidly. This is supported by the observation that cyclin D2 knockout mice are viable with normal hematopoiesis (16). The high throughput screen identified cyproheptadine as an inhibitor of cyclin D2 transactivation, which is over-expressed in patients with high risk AML and MM. Subsequently, the inventors demonstrated that this compound and analogs thereof induce cell death in hematological malignancies including multiple myeloma and leukemia cells.

While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

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4. Trudel S, Li ZH, Rauw J, Tiedemann RE, Wen XY, and Stewart AK. Preclinical studies of the pan-Bel inhibitor obatoclax (GX015-070) in multiple myeloma. Blood 2007;109:5430-8.

5. Sarfaraz S, Afaq F, Adhami VM, Malik A, and Mukhtar H. Cannabinoid receptor agonist induced apoptosis of human prostate cancer cells LNCaP proceeds through sustained activation of ERK1/2 leading to G1 cell cycle arrest. J Biol Chem 2006. 6. Schimmer AD, Hedley DW, Chow S, et al. The BH3 domain of BAD fused to the Antennapedia peptide induces apoptosis via its alpha helical structure and independent of Bcl-2. Cell Death Differ 2001 ;8:725-33.

7. Pham NA, Jacobberger JW, Schimmer AD, Cao P, Gronda M, and Hedley DW. The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice. MoI Cancer Ther 2004;3: 1239-48.

8. Hurt EM, Wiestner A, Rosenwald A, et al. Overexpression of c-maf is a frequent oncogenic event in multiple myeloma that promotes proliferation and pathological interactions with bone marrow stroma. Cancer Cell 2004;5: 191 -9. 9. Rao BS, Das DG, Taraknath VR, and Sarma Y. A double blind controlled study of propranolol and cyproheptadine in migraine prophylaxis. Neurol India 2000;48:223-6.

10. Kardinal CG, Loprinzi CL, Schaid DJ, et al. A controlled trial of cyproheptadine in cancer patients with anorexia and/or cachexia.

Cancer 1990;65:2657-62.

11. Klein GL and Galant SP. A comparison of the antipruritic efficacy of hydroxyzine and cyproheptadine in children with atopic dermatitis. Ann Allergy 1980;44:142-5. 12. Ronchetti D, Finelli P, Richelda R, et al. Molecular analysis of 11q13 breakpoints in multiple myeloma. Blood 1999;93: 1330-7.

13. Mao X, Stewart AK, Hurren R, et al. A chemical biology screen identifies glucocorticoids that regulate c-maf expression by increasing its proteasomal degradation through upregulation of ubiquitin. Blood 2007.

14. Paterson JL, Li Z, Wen XY, et al. Preclinical studies of fibroblast growth factor receptor 3 as a therapeutic target in multiple myeloma. Br J Haematol 2004; 124:595-603.

15. Parada Y, Banerji L, Glassford J, et al. BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression. J Biol Chem 2001 ;276:23572-80.

16. Huang W, Chang HY, Fei T, Wu H, and Chen YG. GSK3 beta mediates suppression of cyclin D2 expression by tumor suppressor PTEN. Oncogene 2007;26:2471-82.

17. Ponzio G, Loubat A, Rochet N, et al. Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction. Oncogene 1998;17:1159-66.

18. Dou QP, An B, and Will PL. Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis. Proc Natl Acad Sci U S A 1995;92:9019-23. 19. Shift SJ, Qiao L, Tsai LL, and Rigas B. Sulindac sulfide, an aspirin-like compound, inhibits proliferation, causes cell cycle quiescence, and induces apoptosis in HT-29 colon adenocarcinoma cells. J Clin Invest 1995;96:491-503. 20. Schimmer A, Hedley DW, Penn LZ, and Minden MD. Receptor- and mitochondrial-mediated apoptosis in acute leukemia: A translational view. Blood 2001 ;98:3541-53.

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Claims

WE CLAIM:

1. A method of treating a proliferative disease involving increased D-cyclin and/or a hematological malignancy comprising administering to a subject in need thereof, an effective amount of a compound selected from a compound of Formula I, and pharmaceutically acceptable salts, solvates and prodrugs thereof:

wherein

^^ is a single or double bond,

R¹ is selected from H, Ci_-4alkyl, C(O)OCi-₄alkyl, C(O)Ci_-4alkyl and

C(O)NHC_1-4alkyl;

R² is selected from H and C-ualkyl; R³ is selected from H and Ci^alkyl; or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring;

R⁴ and R⁵ are independently selected from H, halogen, hydroxy, Ci- ₄alkyl, fluoro-substituted Ci^alkyl and C-i^alkoxy; and X is selected from C and N.

2. A method of treating a hematological malignancy comprising administering to subject in need thereof, an effective amount of a compound selected from a compound of Formula I as defined in claim 1 and pharmaceutically acceptable salts, solvates and prodrugs thereof.

3. The method according to claim 1 or 2, wherein said proliferative disease or hematological malignancy is multiple myeloma.

4. The method according to claim 1 or 2, wherein said proliferative disease or hematological malignancy is leukemia.

5. The method according to claim 4, wherein said leukemia is selected from the group comprising acute myeloid leukemia and acute lymphocytic leukemia.

6. The method according to claim 1 or 2, wherein the proliferative disease or hematological malignancy is lymphoma.

7. The method according to any one of claims 1 to 6, wherein, in the compound of Formula I, R¹ is selected from CH₃ and C(O)OCi-₂alkyl

8. The method according to any one of claims 1-7, wherein, in the compound of Formula I, R² and R³ are independently selected from H and CH₃, or R² and R³ are joined together along with the carbon and nitrogen atoms to which they are attached to form a piperidine ring.

9. The method according to any one of claims 1-7, wherein, in the compound of Formula I₁ R⁴ and R⁵ are independently selected from H,

Cl, F, I, hydroxy, CH₃, CF₃, and and CH₃O.

10. The method according to any one of claims 1-6 wherein the compound of Formula I is selected from cyproheptadene, amitriptyline, cyclobenzaprine, and loratadine, and a pharmaceutically acceptable salt, solvate and prodrug thereof.

11. The method according to claim 10, wherein the compound of Formula I is selected from cyproheptadene, and a pharmaceutically acceptable salt, solvate and prodrug thereof.

12. The method according to any one of claims 1 to 11, wherein said effective amount is about 0.1 to 200 mg/kg body weight.

13. The method according to claim 12, wherein the effective amount is about 0.1 to about 100 mg/kg, about 0.1 to about 80 mg/kg, about 0.1 to about 40 mg/kg, about 0.1 to about 20 mg/kg, about 0.1 to about 2 mg/kg, or about 0.5 to about 2 mg/kg body weight.

14. The method according to any one of claims 1 to 11 , wherein the effective amount is about 2 to about 2000 mg, about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg or about 35 to about 50 mg.

15. Use of a compound of Formula I as defined in any one of claims 1 and 7-11 for treating a proliferative disorder involving increased D-cyclin expression.

16. Use of a compound of Formula I as defined in any one of claims 1 and 7-11 for the preparation of a medicament for the treatment a proliferative disorder involving increased D-cyclin expression.

17. Use of a compound of Formula I as defined in any one of claims 1 and 7-11 for treating a hematological malignancy.

18. Use of a compound of Formula I as defined in any one of claims 1 and 7-11 in the preparation of a medicament for the treatment of a hematological malignancy.

19. The use of any one of claims 15 to 18 wherein the proliferative disorder or hematological malignancy is multiple myeloma.

20. The use of any one of claims 15 to 18 wherein the proliferative disorder or hematological malignancy is leukemia.

21. The use of claim 20 wherein the leukemia is selected from the group consisting of acute myeloid leukemia and acute lymphocytic leukemia.

22. The use of any one of claims 15 to 18 wherein the proliferative disorder or hematological malignancy is lymphoma.

23. The use according to any one of claims 12 to 22, wherein the compound of Formula I is used in an amount that is about 0.1 to 200 mg/kg body weight.

24. The use according to claim 23, wherein the amount is about 0.1 to about 20 mg/kg, about 0.1 to about 2 mg/kg, or about 0.5 to about 2 mg/kg body weight.

25. The use according to any one of claims 12 to 22 wherein compound of Formula I is used in an amount that is about 2 to about 2000 mg, about 10 to about 150 mg, about 10 to about 100 mg, about 20 to about 60 mg or about 35 to about 50 mg.

26. A pharmaceutical composition comprising a compound selected from a compound of Formula I as defined in any one of claims 1 and 7-11 , and a pharmaceutically acceptable salt, solvate and prodrug thereof, and a pharmaceutically acceptable carrier in a solid dosage form that comprises from about 2 to about 2000 mg, about 10 to about 150 mg, about 10 to about 100mg, about 20 to about 60 mg, or about 35 to about

50 mg of the compound.

27. The pharmaceutical composition of claim 26, wherein the dosage form comprises from about 35 to about 50 mg of the compound.

28. A pharmaceutical composition comprising a compound selected from a compound of Formula I as defined in any one of claims 1 and 7-11 , and a pharmaceutically acceptable salt, solvate and prodrug thereof, and a pharmaceutically acceptable carrier in a liquid dosage form that comprises from about 2 to about 2000 mg, about 10 to about 150 mg, about 10 to about 100mg, about 20 to about 60 mg, or about 35 to about 50 mg of the compound.

29. The pharmaceutical composition of claim 28, wherein the dosage form comprises from about 35 to about 50 mg of the compound.

30. A pharmaceutical composition for treatment of acute myeloid leukemia or multiple myeloma in a subject, which composition comprises as active ingredient a compound selected from a compound of Formula I as defined in any one of claims 1 and 7-11, and a pharmaceutically acceptable salt, solvate and prodrug thereof, and a pharmaceutically acceptable carrier in unit dosage form.

31. The pharmaceutical composition of claim 30, wherein the dosage form is for oral administration.

32. The pharmaceutical composition of claim 30, wherein the dosage form is for injection.

33. A pharmaceutical composition comprising a compound selected from a compound of Formula I as defined in any one of claims 1 and 7-11 , and a pharmaceutically acceptable salt, solvate and prodrug thereof, and a pharmaceutically acceptable carrier in unit dosage form in an amount to provide about 0.1 to about 200 mg, about 0.1 to about 100 mg, about 0.1 to about 80 mg, about 0.1 to about 40 mg, about 0.1 to about 20 mg, about 0.1 to about 2 mg, or about 0.5 to about 2 mg of the compound per kg body weight formulated into a solid oral dosage form, a liquid dosage form, or an injectable dosage form.

34. The pharmaceutical composition according to claim 33, wherein the compound is in an amount to provide about 0.1 to about 2 mg of the compound per kg body.

35. The composition as claimed according to any one of claims 29 and 33 to 34 useful for the treatment of a hematological malignancy, the hematological malignancy selected from the group comprising leukemia and multiple myeloma.

36. The composition according to claim 35 wherein the leukemia is acute myeloid leukemia or acute lymphocytic leukemia.

37. A method of identifying D-cyclin modulators comprising: i) treating cells over-expressing a c-maf cassette and a cyclin D promoter driving a reporter cassette with a test compound; ii) treating cells over-expressing a c-maf cassette and a cyclin D promoter driving a reporter cassette without a test compound; iii) comparing reporter cassette activity and cell viability in cells receiving the test compound to cells not receiving the drug wherein a Iog2 ratio of (reporter activity in cells receiving the test compound/ reporter activity in cells not receiving the test compound)/(viability of cells receiving the test compound/viability of cells not receiving the test compound) that is less than -1 is indicative of the test compound being an inhibiting modulator and wherein a ratio of greater than 1 is indicative the test compound being of an activating modulator.