WO2009016488A2

WO2009016488A2 - Compositions comprising tetracyclic antidepressants for treatment of cancer and related methods

Info

Publication number: WO2009016488A2
Application number: PCT/IB2008/002006
Authority: WO
Inventors: Anat Oren; Alex Diber; Sergey Nemzer; Yaron Kinar; Gad S. Cojocaru; Yossi Cohen
Original assignee: Compugen Ltd
Current assignee: Compugen Ltd
Priority date: 2007-08-02
Filing date: 2008-07-30
Publication date: 2009-02-05
Anticipated expiration: 2010-02-02
Also published as: WO2009016488A3

Abstract

A method for treating cancer, for example breast cancer, urologic cancer, glioma, and/or hormone-refractory prostate cancer, and/or a related disorder in a patient comprises administering HTR2A antagonists to the patient.

Description

95798-258468

COMPOSITIONS COMPRISING TETRACYCLIC ANTIDEPRESSANTS FOR TREATMENT OF CANCER AND RELATED METHODS

FIELD OF THE INVENTION

[0001] The present invention relates to the use of HTR2A antagonists for treatment of cancer, for example breast tumors, gliomas, ho rmone- refractory prostate cancer and urologic tumors, particularly bladder tumors.

BACKGROUND OF THE INVENTION

[0002] Breast cancer is a malignancy of the breast tissue, including of the ducts, connective tissue and/or epithelial tissue, although it may present in the lymph nodes and even throughout the body (due to metastasis of the original lesion). Although it is more common in women, breast cancer may occur in men as well. (Robson M, Offit K. Clinical practice. Management of an inherited predisposition to breast cancer. N Engl J Med. 2007 JuI 12;357(2): 154-62; Harman SM. Estrogen replacement in menopausal women: recent and current prospective studies, the WHI and the KEEPS. Gend Med. 2006 Dec;3(4):254-69; Pusztai L, Cristofanilli M, Paik S. New generation of molecular prognostic and predictive tests for breast cancer. Semin Oncol. 2007 Apr;34(2 Suppl 3):S10-6.)

[0003] Chemo therapeutic options for breast cancer include retinoids; cyclooxygenase

(COX) inhibitors; selective estrogen receptor modulators (SERM) such as tamoxifen; aromatase inhibitors such as anastrozole, letrozole, and exemestane; anthracyclines; taxanes; cyclophosphamide; and trastuzumab. However, many breast cancers prove to be refractory to such treatments and/or may be diagnosed too late to benefit from these treatments, particularly since many treatments are most effective for earlier stage cancers.

[0004] Bladder cancer is a common urologic cancer. The most common type of bladder cancer in the United States is urothelial carcinoma, formerly known as transitional cell carcinoma (TCC). The urothelium in the entire urinary tract may be involved, including the renal pelvis, ureter, bladder, and urethra. (Wang X, MacLennan GT, Lopez-Beltran A, Cheng L. Small cell carcinoma of the urinary bladdcr-histogenesis, genetics, diagnosis, biomarkers, treatment, and prognosis. Appl lmmunohistochem MoI Morphol. 2007 Mar;15(l):8-18; Josephson D, Pasin E, Stein JP. Superficial bladder cancer: part 2. Management. Expert Rev Anticancer Ther. 2007 Apr;7(4):567-81; Clark PE. Bladder cancer. Curr Opin Oncol. 2007 May;19(3):241-7.) [0005] Various treatments are known in the art for bladder cancer, including both surgical and pharmaceutical approaches. Examples of such treatments include Bacillus Calmette-Guerin (BCG) immunotherapy, interferon alpha or gamma, valrubicin, intravesical triethylenethiophosphoramide (thiotepa [Thioplex]), mitomycin-C, doxorubicin, and epirubicin, MVAC (a combination therapy of methotrexate, vinblastine, doxorubicin (i.e., Adriamycin) and cisplatin), GC (a combination therapy of gemcitabine and cisplatin), ifosfamide, paclitaxel, docetaxel, oxaliplatin and carboplatin.

[0006] However, many bladder cancers prove to be refractory to such treatments and/or may be diagnosed too late to benefit from these treatments, particularly since many treatments are most effective for earlier stage cancers.

[0007] Glioblastoma multiforme (GBM) is the most common and most aggressive of the primary brain tumors. The current World Health Organization (WHO) classification of primary brain tumors lists GBM as a grade IV astrocytoma. It is highly malignant, infiltrates the brain extensively, and at times may become enormous before turning symptomatic. (Miller CR, Perry A. Glioblastoma. Arch Pathol Lab Med. 2007 Mar;131(3):397-406; Kim L, Glantz M. Chemotherapeutic options for primary brain tumors. Curr Treat Options Oncol. 2006 Nov;7(6):467-78; Sathornsumetee S, Rich JN. New treatment strategies for malignant gliomas. Expert Rev Anticancer Ther. 2006 Jul;6(7): 1087- 104.)

[0008] In GBM, surgery is typically performed yet cannot remove all of the tumor, since it is a highly infiltrating tumor. After surgery, radiation therapy remains the most effective adjuvant therapy for the treatment of patients with HGA/GBM. Radiotherapy prolongs the median survival by 14-36 weeks. Different methods of administering radiation therapy are available, including external beam radiation, stereotactic brachytherapy, stereotactic radiosurgery, and boron neutron capture therapy. Chemotherapy may be used in combination with radiation therapy after surgery, although its effects are limited. Other therapeutic options include 1,3- bis(2-chloroethyl)- 1 -nitrosourea (BCNU), 1 -(2-chloroethyl)-3-cyclohexyl- 1 -nitrosourea (CCNU), cisplatin, carboplatin, oxaliplatin and etoposide. However their efficacy is limited. [0009] Extensive research is taking place on newer therapeutic options which are unfortunately not yet available, so their efficacy is unknown. Thus clearly new treatments for gliomas are urgently needed. [0010] Given the current difficulty of successfully treating breast and bladder cancers, and gliomas, as well as the likelihood of recurrence, and the fact that many patients do not respond to available treatments or alternatively respond initially but then stop responding, there is an unmet need to develop therapies which can successfully treat these cancers.

SUMMARY OF THE INVENTION

[0011] The present invention overcomes the disadvantages of preexisting treatment options by providing a method for treating breast and urologic cancers, hormone-refractory prostate cancer and gliomas, with 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A) HTR2A antagonists. Without wishing to be limited by a single hypothesis, it is believed that the HTR2A antagonists operate through a different mechanism of action than available treatments, and hence may be effective for patients with initial cancer lesions, patients who do not respond to such available treatments, or who initially respond but then stop responding, and/or for recurrent breast and urologic cancers, hormone-refractory prostate cancer and gliomas. [0012] In one aspect, the invention provides methods for treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a composition comprising an HTR2A antagonist, thereby treating the cancer. The cancer can be, without limitation, breast cancer, urologic cancer, hormone-refractory prostate cancer, and a glioma. The HTR2A antagonist can be, without limitation, mianserin, amoxapine, maprotiline, mirtazapine, trazodone, or combinations thereof. The compositions can be administered to the patients via any method known in the ait, including, without limitation, orally, rectally, transmucosally, transnasally, intestinally, parenterally, intramuscularly, subcutaneously, intramedullarily, intrathecally, intraventricularly, intravenously, intraperitoneally, intranasally, intraocularly, or transurethrally.

[0013] In some embodiments, the methods further comprise administering a secondary therapeutic agent. Thus, treatment of diseases using the composition of the present invention may be in combination therapy with at least one other therapeutic or immune modulatory agent, including, but not limited to, chemotherapeutic agents such as cytotoxic and cytostatic agents; immunological modifiers such as cytokines; folic acid; vitamins; minerals; aromatase inhibitors; RNAi; Histone Deacetylase Inhibitors; proteasome inhibitors; GNRH (gonadotropin releasing factor)-antagonists; antibody therapy agents; and/or anti-hormone therapy agents, such as estrogen analogs. The secondary therapeutic agent can be selected from, without limitation, retinoids; cyclooxygenase (COX) inhibitors; estrogen receptor modulators such as, for example, tamoxifen, toremifene and fulvestrant; aromatase inhibitors such as, for example, anastrozole, letrozole, and exemestane; trastuzumab; anti-hormone agents; MVAC; platin-based chemotherapeutic agents such as, for example, carboplatin, oxaliplatin, and cisplatin; taxol; taxotere; gemcitabine; GC (the combination of gemcitabine and cisplatin); temozolomide; BCNU; CCNU; etoposide; leuprolide; goserelin; piOgesterone-like drugs such as, for example, megestrol acetate; or combinations thereof. For example, the HTR2A antagonist can be mianserin and the secondary threrapeutic agent can be tamoxifen. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be a platin-based chemotherapeutic agent. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be carboplatin. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be cisplatin. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be oxaliplatin. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be toremifene. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be fulvestrant. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be letrozole. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be anastrozole. The HTR2A antagonist can also be administered as part of a cocktail comprising any of the above combination therapies, and further comprising other chemotherapeutic agents, including chemotherapeutic regimes such as, for example, such as docetaxel plus doxorubicin and cyclophosphamide (TAC) or docetaxel and fluorouracil plus doxorubicin and cyclophosphamide (FAC). The secondary therapeutic agent can be administered prior to, after, or simultaneously with administration of the HTR2A antagonist.

[0014] In some embodiments, the methods of the present invention comprise administering any of the pharmaceutical compositions of the present invention, including those comprising an HTR2A antagonist as well as those also comprising a secondary therapeutic agent, without diagnosing depression in the patient. In some embodiments, the patient does not exhibit clinical symptoms of depression. The pharmaceutical composition can, for example, be administered daily for a period of about 1 month to about 3 months, or for a period of at least about 1 to at least about 1 year, or longer, as would be appreciated by a person of skill in the art. [0015] In some embodiments, the methods of the present invention further comprise performing a secondary procedure. The secondary procedure can be selected from, without limitation, radiation treatment, surgery or a combination thereof. The secondary procedure can also be, for example, ovarian ablation. The secondary procedure can be performed prior to, after, or simultaneously with administration of the HTR2A antagonist.

[0016] In another aspect, the invention provides one or more pharmaceutical compositions comprising an HTR2A antagonist, including a salt or derivative, in a therapeutically effective amount to treat cancer. In some embodiments, the cancer that is treated can be, without limitation, breast cancer, urologic cancer, hormone-refractory prostate cancer or a glioma. In some embodiments, the HTR2A antagonist can be, without limitation, mianserin, amoxapine, maprotiline, mirtazapine, trazodone, or combinations thereof. In some embodiments, the pharmaceutical compositions comprise a secondary therapeutic agent. In some embodiments, the pharmaceutical compositions comprise a combination therapy comprising a tetracyclic antidepressant and a secondary therapeutic agent, wherein the secondary therapeutic agent has a first effective dosage for treating cancer when administered alone, and the dosage in the pharmaceutical composition is lower than the first effective dosage of the secondary therapeutic agent, the combination therapy achieving at least substantially the same efficacy in inhibiting cancer cell growth as the secondary therapeutic agent achieves when administered alone in the first effective dosage.

[0017] In another aspect, the invention provides kits comprising a therapeutically effective amount of an HTR2A antagonist such as a tetracyclic antidepressant for the treatment of cancer, a container, and a label associated with the container, wherein the label contains directions for administration of the tetracyclic antidepressant for treatment of cancer. In some embodiments, the container is a blister pack. The tetracyclic antidepressant can be mianserin. The kits can also comprise a pharmaceutical composition comprising a secondary therapeutic agent. In some embodiments, the label provides directions for administering the tetracyclic antidepressant and secondary therapeutic agent for treatment of cancer.

[0018] In another aspect, the invention provides methods of diagnosing cancer that is responsive to an HTR2A antagonist. In some embodiments, the methods comprise measuring HTR2A activity in a tissue, and identifying tissue having elevated HTR2A activity as potentially cancerous. In some embodiments, measuring HTR2A activity comprises measuring expression of HTR2A protein or measuring ability to bind serotonin in cells of a patient. [0019] In another aspect, the invention provides methods of assaying activity of a compound of interest. In some embodiments, the methods comprise measuring HTR2A activity in a cancer cell line, contacting the cancer cell line with a compound of interest, and determining if the compound reduces HTR2A activity in the cancer cell line. In some embodiments, the methods also comprise contacting the cancer cell line with a secondary therapeutic agent, and determining if the secondary therapeutic agent further reduces HTR2A activity in the cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 presents a comparison of HTR2A expression levels in human breast cancer cell lines. HTR2A expression was determined in cell lysates by immunoblotting with an anti~HTR2A antibody (upper panel), and reprobing with anti-tubulin antibody (middle panel). HTR2A expression levels detected in the immunoblot were normalized to tubulin protein levels (lower panel).

[0021] Figure 2 presents a series of graphs demonstrating the inhibition of cell proliferation in several types of cancer cells by mianserin.

[0022] Figure 2A depicts the inhibition of cell proliferation in MCF7 cells by mianserin.

MCF7 cells, grown in medium containing 0.1% FBS, were treated with the indicated concentrations of mianserin. Cell proliferation was measured after 72 hrs using BrdU incorporation assay. Results are shown as relative growth compared to untreated cells.

[0023] Figure 2B depicts the inhibition of cell proliferation in MDA-MB-468 cells by mianserin.

[0024] Figure 2C depicts the inhibition of cell proliferation in T98G cells by mianserin.

[0025] Figure 2D depicts the inhibition of cell proliferation in U87-MG cells by mianserin. [0026] Figure 2E depicts the inhibition of cell proliferation in SCaBER cells by mianserin.

[0027] Figure 2F is a graph depicting the effect of mianserin in combination with

Carboplatin on proliferation of MCF7 cells.

[0028] Figure 2G: is a graph depicting the effect of mianserin in combination with

Tamoxifen on proliferation of MCF7 cells.

[0029] Figure 3 is a graph depicting tumor volume after treatment with mianserin (MIA) and / or tamoxifen (TAM) in balb/c nude mice.

DETAILED DESCRIPTION

[0030] Embodiments of the invention are discussed in detail below, using specific terminology for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention. All references cited herein are incorporated by reference as if each had been individually incoiporated.

[0031] The patient according to the present invention is a mammal, such as a human, which is diagnosed with one of the diseases, disorders or conditions described herein, or alternatively is predisposed to at least one type of the diseases, disorders or conditions described herein. The compositions of the present invention can be administered to any mammal in need of the composition that can experience the beneficial effects of the compounds of the invention. Any such mammal is considered a "patient." Such patients include humans and non-humans, such as humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. The patient can be a man or a woman.

[0032] "Treat" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting at least one of the symptoms or deleterious effects of the diseases, disorders or conditions described herein. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Hence, the patient to be treated herein may have been diagnosed as having the disorder or may be predisposed or Susceptible to the disorder.

[0033] A "disorder" is any condition that would benefit from treatment with the agent according to the present invention. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. These disorders include cancer, for example breast and urologic cancers, hormone-refractory prostate cancer as well as gliomas, and related disorders as described herein. [0034] Herein the term "active ingredient" refers to the preparation accountable for the biological effect. An active ingredient may be referred to as an "agent" or an "active agent." As used herein, "active ingredient" encompasses, but is not limited to, HTR2A antagonists and secondary therapeutic agents.

[0035] The term "therapeutically effective amount" refers to an amount of an HTR2A antagonist (and optionally one or more other agents as described herein) that is effective to treat breast and/or urologic cancers, and/or gliomas, and/or hormone-refractory prostate cancer and/or a related disease or disorder in a mammal. Without wishing to be limited, the therapeutically effective amount of the agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and/or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and/or stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. For example, the therapeutically effective amount of the HTR2A antagonist may be up to or at least about 5, 10, 25, 50, 100 mg/kg/day or more. For example, the therapeutically effective amount of the HTR2A antagonist may be up to or at least about 100 mg, 250 mg, 500 mg, Ig, 2g, 4g or more per day. The therapeutically effective amount of the HTR2A antagonist may be different if a secondary therapeutic agent is included in the therapeutic regimen. The therapeutically effective amount of the HTR2A antagonist under these circumstances may readily be determined by a practitioner using animal and clinical trials and medical observations. To the extent that HTR2A antagonists (or another agent) may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR). [0036] As used herein, "estrogen receptor modulator" and "selective estrogen receptor modulator" are substantially equivalent terms.

Compositions

[0037] In one aspect, the invention provides one or more compositions comprising an

HTR2A antagonist or a salt or derivative thereof in a therapeutically effective amount to treat cancer.

[0038] The 5-HT 2A receptor is a subtype of the 5-HT2 receptor. It belongs to the serotonin receptor family and is a G protein coupled receptor (GPCR). HTR2A is the main excitatory receptor subtype among the GPCRs for serotonin (5-hydroxytryptamine or 5-HT), although 5HTR2A may also have an inhibitory effect in certain brain areas. As used herein, "5- HT 2A receptor" and "HTR2A" are substantially equivalent terms.

[0039] As used herein, "HTR2A antagonist" includes any compound that modulates the

HTR2A's ability to bind serotonin and is effective in treating cancers such as breast cancer, urologic cancer, gliomas, and hormone-refractory prostate cancer. In some embodiments, the HTR2A antagonist can be a tetracyclic antidepressant, including, without limitation, mianserin, amoxapine, maprotiline, mirtazapine, trazodone, or combinations thereof. In some embodiments, the HTR2A antagonist can be, without limitation, volinanserin, ziprasidone, iloperidone depot, trelanserin, ITI-007 (Bristol-Myers Squibb Co.), ITI-722 (Bristol-Myers Squibb Co.), iloperidone, eplivanserin, asenapine, BVT-28949 (Biovitrum AB), ocaperidone, 773812 (GlaxoSmithKline PLC), AVE-8488 (Sanofi Aventis), S-35120 (Servier), aripiprazole, pruvanserin, flibanserin, QF-2004B (Universidade de Santiago de Compostela), R-167154 (Johnson & Johnson), abaperidone, Org-50081 (Organon), ICI- 169369 (Zeneca Group PLC), IT- 657 (DuPont Pharmaceuticals), LEK-8829 (Lek Pharmaceutical and Chemical Co., DD), deramciclane, amperozide, F-97013-GD (FAES Farma SA), LY-367265 (Eli Lilly & Co.), LY- 433221 (Eli Lilly & Co.), SEP-89406 (Sepracor Inc.), ketanserin analogs, FG-5938 (Nerviano), FG-5974 (Nerviano), RP-71602 (Rhone-Poulenc SA), S-14956 (Servier), S-16924 (Rhone- Poulenc SA), S-17828 (Servier), ZD-3638 (AstraZeneca), JL-13 (Therabel Research), nantenine, anxiolytics, Adir, gamma-mangostin, TY-1 1223 (Boehringer Ingelheim International GmbH) and S-21357-1 (Servier). The term "HTR2A antagonist" also encompasses dopamine D2/serotonin 2A antagonists, serotonin antagonists, antipsychotics, antipsychotic therapeutics, broad-spectrum antipsychotics, atypical antipsychotics, dopamine D4 antagonists that are effective according to the present invention. The term "HTR2A antagonist" also encompasses any salts, prodrugs, metabolites, analogues, derivatives and polymorphs of any of the aforementioned HTR2A antagonists having the desired activity. As used herein, "HTR2A receptor antagonist" and "HTR2A antagonist" are substantially equivalent terms. The term "HTR2A antagonist" also encompasses compounds with other activities, such as dopamine D2/serotonin 2A antagonists, serotonin antagonists, antipsychotics, antipsychotic therapeutics, broad-spectrum antipsychotics, atypical antipsychotics, and dopamine D4 antagonists that are effective according to the present invention. The term "HTR2A antagonist" also encompasses any salts, prodrugs, metabolites, analogues, derivatives and polymorphs of any of the aforementioned HTR2A antagonists having the desired activity. As used herein, "HTR2A receptor antagonist" and "HTR2A antagonist" are substantially equivalent terms. [0040] In some embodiments, the HTR2A antagonist is mianserin. Mianserin is a tetracyclic antidepressant that has antihistaminic and hypnosedative, but almost no anticholinergic, effects. Mianserin is a weak inhibitor of norepinephrine reuptake and strongly stimulates the release of norepinephrine. Interactions with serotonin receptors in the central nervous system have also been found. Its effect is usually noticeable after one to three weeks. [0041] HTR2A antagonists can be provided to the subject per se, or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable earner, optionally with one or more other agents as described herein. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0042] In some embodiments, the inventive compositions further comprise a secondary therapeutic agent. As used herein, "secondary therapeutic agent" refers to any agent that can be administered simultaneously with, prior to or subsequently to the HTR2A antagonist in such a way as to be therapeutically effective according to the present invention. The secondary therapeutic agent can be selected from, without limitation, retinoids, cyclooxygenase (COX) •inhibitors, estrogen receptor modulators, such as tamoxifen, toremifene, fulvestrant; aromatase inhibitors, such as anastrozole, letrozole, and exemestane; trastuzumab; MVAC; platin-based chemothcrapeutic agents, such as carboplatin, oxaliplatin, and cisplatin; taxol; taxotere; gemcitabine; GC (the combination of gemcitabine and cisplatin); temozolomide; BCNU; CCNU; etoposide; leuprolide; goserelin; progesterone-like drugs such as megestrol acetate; or combinations thereof. The term "secondary therapeutic agent" also encompasses any salts, prodrugs, metabolites, analogues, derivatives and polymorphs of any of the aforementioned secondary therapeutic agents. As used herein, "secondary treatment" encompasses the terms "secondary therapeutic agent" and "secondary procedure."

[0043] For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be tamoxifen. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be carboplatin. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be cisplatin. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be oxaliplatin. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be toremifene. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be fulvestrant. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be letrozole. For example, the HTR2A antagonist can be mianserin and the secondary therapeutic agent can be anastrozole. In any of the aforementioned combination therapies, another tetracyclic antidepressant can be substituted for mianserin. For example, mianserin can be replaced with amoxapine, maprotiline, mirtazapine, trazodone, or another tetracyclic antidepressant in any of these, or any other, combination therapy according to the present invention.

[0044] The combination therapies comprising an HTR2A antagonist exhibit several advantages. For example, administering a combination therapy may permit the administration of a lower dosage of the secondary therapeutic agent than would be used if the secondary therapeutic agent were being administered alone, while still maintaining the at least substantially the same therapeutic effectiveness. For example, a combination therapy comprising an HTR2A antagonist and a secondary therapeutic agent according to the present invention may permit the health care provider to reduce the dosage of the secondary therapeutic agent to 1/10 (i.e., one- tenth) of its individual-use dosage, while still achieving at least substantially the same therapeutic effectiveness. In this way, the severity of side effects can be reduced, which can allow the patient to remain on the treatment regimen for longer periods. In addition, for patients who are unable to tolerate the individual dosage level of the secondary therapeutic agent, the reduction in dosage level achieved by a combination therapy may permit them to undergo treatment involving the secondary therapeutic agent when it may not have been possible otherwise.

[0045] The secondary treatment may optionally include any treatment protocol for breast cancer, including but not limited to retinoids, cyclooxygenase (COX) inhibitors, selective estrogen receptor modulators (SERM) such as tamoxifen; aromatase inhibitors (AI), including but not limited to anastrozole, letrozole and exemestane; and Trastuzumab.

[0046] The secondary treatment may optionally include any treatment protocol for bladder cancer, including but not limited to MVAC (cisplatin, methotrexate, doxorubicin and vinblastine), carboplatin, oxaliplatin, taxol, taxotere, gemcitabine, cisplatin and the combination of gemcitabine and cisplatin.

[0047] The secondary treatment may optionally include any treatment protocol for gliomas, including but not limited to radiation treatment, temozolomide, nitrosoureas, BCNU and CCNU, and chemotherapies such as cisplatin, carboplatin, oxaliplatin and etoposide.

[0048] The secondary treatment may optionally include any treatment protocol for hormone-refractory prostate cancer, including but not limited to docetaxel alone or in combination with estramustine, mitoxantrone in combination with prednisone, taxan based chemotherapy, and tamoxifen.

[0049] For any of the above cancers, the secondary treatment may optionally include surgery or radiation therapy.

[0050] The compositions and methods of the present invention can be used for the treatment of breast and urologic cancers, hormone-refractory prostate cancer, as well as gliomas, or a related disease, disorder or condition.

[0051] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0052] Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0053] As used herein, the phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier," which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. As used herein, "carrier" encompasses any adjuvant that can be used in the treatment of cancer. One of the ingredients included in the pharmaceutically acceptable earner can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979). These may include water, ethanol, or any other suitable liquid or solid material.

[0054] The term "excipient" includes an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. [0055] Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Methods

[0056] In another aspect, the invention provides methods for treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of any of the compositions of the present invention, including those comprising a secondary therapeutic agent, to the patient, thereby treating the cancer. Thus, according to an additional aspect of the present invention there is provided a method of treating a breast and urologic cancers, hormone- refractory prostate cancer, as well as gliomas, or a related disease, disorder or condition in a subject.

[0057] For example, the methods of the present invention can comprise administration of mianserin to a cancer patient for the treatment of cancers such as breast and urologic cancers, hormone-refractory prostate cancer, and gliomas, either individually or in combination with a secondary treatment. Mianserin can be administered orally to a patient. However, the present invention contemplates administration of mianserin via any route that is consistent with effective treatment of cancer, as determined by a person of skill in the art. The therapeutically effective dosage of mianserin for the treatment of cancer can be determined by a person of skill in the art. Such a dosage can be, for example, up to or at least about 1 mg/day, about 5 mg/day, about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/ day, about 100 mg/day, about 110 mg/day, about 120 mg/day, about 200 mg/day, about 300 mg/day or more. As used herein, "about" may refer to a range from 10% below the referenced number to 10% above the referenced number. For example, "about 50" may mean from 45 to 55. Other meanings of "about" may be apparent from the context.

[0058] For example, the methods of the present invention can comprise administration of amoxapine to a cancer patient for the treatment of cancers such as breast and urologic cancers, hormone-refractory prostate cancer, and gliomas, either individually or in combination with a secondary treatment. Amoxapine can be administered orally to a patient. However, the present invention contemplates administration of amoxapine via any route that is consistent with effective treatment of cancer, as determined by a person of skill in the art. The therapeutically effective dosage of amoxapine for the treatment of cancer can be determined by a person of skill in the art. Such a dosage can be, for example, up to or at least about 1 mg/day, about 5 mg/day, about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/ day, about 100 mg/day, about 110 mg/day, about 120 mg/day, about 150 mg/day, about 200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day, about 400 mg/day, about 450 mg/day, about 500 mg/day, about 550 mg/day, about 600 mg/day, about 700 mg/day, about 1000 mg/day, or more.

[0059] For example, the methods of the present invention can comprise administration of maprotiline to a cancer patient for the treatment of cancers such as breast and urologic cancers, hormone-refractory prostate cancer, and gliomas, either individually or in combination with a secondary treatment. Maprotiline can be administered orally to a patient. However, the present invention contemplates administration of maprotiline via any route that is consistent with effective treatment of cancer, as determined by a person of skill in the art. The therapeutically effective dosage of maprotiline for the treatment of cancer can be determined by a person of skill in the art. Such a dosage can be, for example, up to or at least about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 55 mg/day, about 60 mg/day, about 65 mg/day, about 70 mg/day, about 75 mg/day, about 80 mg/day, about 90 mg/ day, about 100 mg/day, about 1 10 mg/day, about 120 mg/day, about 150 mg/day, about 200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day, about 400 mg/day, or more. [0060] For example, the methods of the present invention can comprise administration of mirtazapine to a cancer patient for the treatment of cancers such as breast and urologic cancers, hormone-refractory prostate cancer, and gliomas, either individually or in combination with a secondary treatment. Mirtazapine can be administered orally to a patient. However, the present invention contemplates administration of mirtazapine via any route that is consistent with effective treatment of cancer, as determined by a person of skill in the art. The therapeutically effective dosage of mirtazapine for the treatment of cancer can be determined by a person of skill in the art. Such a dosage can be, for example, up to or at least about 1 mg/day, about 5 mg/day, about 10 mg/day, about 12.5 mg/day, about 15 mg/day, about 17.5 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/ day, about 100 mg/day, about 110 mg/day, about 120 mg/day, about 150 mg/day, about 200 mg/day, or more. [0061] For example, the methods of the present invention can comprise administration of trazodone to a cancer patient for the treatment of cancers such as breast and urologic cancers, hormone-refractory prostate cancer, and gliomas, either individually or in combination with a secondary treatment. Trazodone can be administered orally to a patient. However, the present invention contemplates administration of trazodone via any route that is consistent with effective treatment of cancer, as determined by a person of skill in the art. The therapeutically effective dosage of trazodone for the treatment of cancer can be determined by a person of skill in the art. Such a dosage can be, for example, up to or at least about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 55 mg/day, about 60 mg/day, about 65 mg/day, about 70 mg/day, about 75 mg/day, about 80 mg/day, about 90 mg/ day, about 100 mg/day, about 110 mg/day, about 120 mg/day, about 150 mg/day, about 200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day, about 400 mg/day, about 450 mg/day, about 500 mg/day, about 550 mg/day, about 600 mg/day, about 700 mg/day, about 1000 mg/day, or more. The daily dosage of trazodone can be administered in several unit dosages daily. For example, the daily dosage can be administered in three unit dosages.

[0062] In some embodiments, the methods of the present invention can comprise administration of a secondary therapeutic agent. For example, the secondary therapeutic agent can be tamoxifen. Tamoxifen can be administered orally to a patient. However, the present invention contemplates administration of tamoxifen via any route that is consistent with effective treatment of cancer, as determined by a person of skill in the art. The therapeutically effective dosage of tamoxifen for the treatment of cancer can be determined by a person of skill in the art. Such a dosage can be, for example, up to or at least about 1 mg/day, about 5 mg/day, about 10 mg/day, about 12.5 mg/day, about 15 mg/day, about 17.5 mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 60 mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/ day, about 100 mg/day, about 110 mg/day, about 120 mg/day, about 150 mg/day, about 200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day, about 400 mg/day, about 450 mg/day, about 500 mg/day, about 550 mg/day, about 600 mg/day, about 700 mg/day, about 1000 mg/day, or more. [0063] In some embodiments, the compositions and methods of the present invention can be used to treat breast cancer. As used herein, "breast cancer" encompasses, without limitation, carcinoma of the breast, cancer of the breast, adenocarcinoma of the breast, infiltrating ductal carcinoma, invasive ductal carcinoma, infiltrating lobular carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, DCIS, lobular carcinoma in situ, LCIS, Li-Fraumeni syndrome, Cowden disease, basal-like breast cancer and Muir-Torre syndrome.

[0064] Approximately 5-10% of breast cancer cases are primarily attributable to inherited factors. More controversial risk factors include obesity, low-dose radiation, and oral contraceptive use. The Breast Cancer Detection Demonstration Project (BCDDP) defined several factors not associated with increased risk, including oral contraceptive use, long-term (greater than 15 years) menopausal estrogen use, modest alcohol use, and cigarette smoking. Various analyses of data from the Nurses' Health Study also have shown no association between oral contraceptive use and breast cancer. Conflicting data preclude the recommendation of guidelines for risk modification in these areas.

[0065] Epithelial tumors comprise the largest group, including intraductal papilloma, adenomas, intraductal and lobular carcinoma in situ, invasive (ductal and lobular) carcinoma, and Paget disease of the nipple. Invasive ductal carcinoma is by far the most common type. [0066] Phyllodes tumor, benign and malignant, and carcinosarcoma are rare lesions grouped as mixed connective tissue and epithelial tumors. Other common nonadenocarcinoma lesions of the breast include angiosarcoma and primary lymphoma.

[0067] Invasiveness is a key determinant in the prognosis and treatment of breast malignancy. Noninvasive lesions are by definition limited by the basement membrane and may be classified as DCIS (ductal carcinoma in situ) or LCIS (lobular carcinoma in situ). DCIS is by far more common than LCIS, and more importantly, it should be distinguished as a clearly malignant lesion; LCIS, by contrast is itself benign although it may indicate an increased chance of breast cancer in the future. Ductal epithelial cells undergo malignant transformation and proliferate intraluminally. Eventually, the cells outstrip their blood supply and become necrotic centrally. This debris can calcify and be detected mammographically. Moreover, the lesions also may be palpable clinically. Five pathologic subtypes have been identified: comedo, papillary, micropapillary, solid, and cribriform. Most lesions represent a combination of at least two of these subtypes. The presence of comedo necrosis is an independent risk factor for subsequent ipsilateral breast cancer (NSABP-B 17).

[0068] LCIS also arises from epithelial cells; however, their growth continues in a lobular pattern. In contrast to DCIS, these lesions rarely develop central necrosis, calcify, or become palpable. For this reason, LCIS rarely is detected by examination or mammography preoperatively and usually presents as an incidental finding on histologic review. Clinically, LCIS is considered more of a marker for future development of invasive cancer rather than a malignant lesion. The risk of subsequent breast cancer is equal for both breasts irrespective of the index site. Moreover, most invasive cancers that develop are infiltrating ductal cancers, which support the concept that LCIS is not a malignant lesion.

[0069] Invasive breast cancers usually are epithelial tumors of ductal or lobular origin.

Features such as size, status of surgical margin, estrogen receptors (ER) and progesterone receptors (PR), nuclear and histologic grade, DNA content, S-phase fraction, vascular invasion, tumor necrosis, and quantity of intraductal component are all important in deciding on a course of treatment for any breast tumor.

[0070] Medical therapy for breast cancer can be divided into 3 categories: chemoprevention, neo-adjuvant, and adjuvant therapy. All can be considered to be a secondary treatment with an HTR2A antagonist according to the present invention. With regard to chemoprevention, several classes of drugs, including retinoids, cyclooxygenase (COX) inhibitors, and selective estrogen receptor modulators (SERM), have been studied in the chemoprevention of breast cancer. The NSABP-Pl trial has demonstrated the efficacy of tamoxifen, a SERM, in the prevention of invasive breast cancer. Treatment with tamoxifen reduced the risk of invasive breast cancer by 49%. Moreover, a 50% reduction in the risk of noninvasive cancers was demonstrated. In terms of prevention, tamoxifen is approved by the US Food and Drug Administration (FDA) for use in healthy women at high risk for the development of invasive breast cancer and in patients with early invasive lesions at risk of secondary contralateral cancer. Dosing should be 20 mg/d for 5 years. Significant relative toxicity from tamoxifen use includes increased risk of endometrial cancer and pulmonary embolism in women older than 50 years. Currently, the NSABP P-2 trial (Study of Tamoxifen and Raloxifene [STAR] trial) is underway to compare the efficacy of tamoxifen with raloxifene, which is a SERM used for treatment of osteoporosis.

[0071] A relatively new class of drugs, aromatase inhibitors (AI), is being explored for efficacy in the chemoprevention of breast cancer. Three third-generation aromatase inhibitors (anastrozole, letrozole, exemestane) have already been approved by the FDA for use in adjuvant therapy for postmenopausal women with hormone receptor-positive breast cancer. [0072] However, tamoxifen remains the standard of care for adjuvant endocrine therapy for premenopausal women. In select patients with small tumors and no nodal metastases, tamoxifen may provide a means for systemic treatment without the use of systemic chemotherapy. Conversely, ER(-) tumors are predictive of improved response to chemotherapy and minimal benefit from tamoxifen.

[0073] Trastuzumab is a relatively new chimerized mouse/human monoclonal antibody that targets the extracellular portion of the Her-2/neu membrane protein. Two major research studies, NSABP B-31 and North Central Cancer Treatment Group (NCCTG) trial N9831, were closed early because of clinically significant findings of improved disease-free and overall survival in women treated with trastuzumab in addition to chemotherapy. Trastuzumab, in combination with chemotherapy, is now recommended to all patients who have lymph node- positive Her-2-positive breast cancer, unless a clear contraindication exists to treatment. Consideration should be made for the significant cardiotoxicity associated with trastuzumab. [0074] Neoadjuvant therapy for locally advanced breast cancer should involve anthracycline or taxane-based regimens.

[0075] Various chemotherapeutic regimens used following surgical treatment include anthracyclines, taxanes, and cyclophosphamide. Choice of the optimal adjuvant chemotherapeutic regimen should be made based on multiple factors including patient age, menopausal status, hormone receptor and Her-2/neu expression, lymph node involvement, and size of the primary lesion.

[0076] In some embodiments, the compositions and methods of the present invention can be used to treat urologic cancer. "Urologic cancer" encompasses, without limitation, urothelial cancer, transitional cell cancer, transitional cell carcinoma, TCC, urothelial tumors, carcinoma in situ, CIS, squamous cell carcinoma and/or SCC when present in the urological system, urothelial carcinoma and bladder cancer. Any part of the urothelium in the urinary tract may be involved, including any one or more of the renal pelvis, ureter, bladder, and urethra. Also encompassed are metastatic cancers having their origin in any of the above cancers and/or locations. Hereinafter, the term "urologic cancer" includes all of these different cancers unless otherwise indicated.

[0077] The clinical course of bladder cancer varies widely among patients with respect to aggressiveness and risk. Low-grade, superficial bladder cancers have minimal risk of progression to death; however, high-grade muscle-invasive cancers are often lethal.

[0078] Almost all bladder cancers are epithelial in origin. The urothelium consists of a 3- to 7-cell mucosal layer within the muscular bladder. Of these urothelial tumors, more than 90% are transitional cell carcinomas. However, up to 5% of bladder cancers are squamous cell in origin, and 2% are adenocarcinomas. Non-urothelial primary bladder tumors are extremely rare and may include small cell carcinoma, carcinosarcoma, primary lymphoma, and sarcoma. [0079] Bladder cancer is often characterized as a polyclonal field change defect with frequent recurrences due to a heightened potential for malignant transformation. However, bladder cancer has also been described as a problem with implantation and migration from a previously affected site.

[0080] The World Health Organization classifies bladder cancers as low grade (grade 1 and 2) or high grade (grade 3). Tumors are also classified by growth patterns: papillary (70%), sessile or mixed (20%), and nodular (10%). Carcinoma in situ (CIS) is a flat, noninvasive, high- grade urothelial carcinoma. The most significant prognostic factors for bladder cancer are grade, depth of invasion, and the presence of CIS.

[0081] Upon presentation, 55-60% of patients have low-grade superficial disease, which is usually treated conservatively with transurethral resection and periodic cystoscopy. Forty to forty-five percent of patients have high-grade disease, of which 50% is muscle invasive and is typically treated with radical cystectomy.

[0082] Less than 5% of bladder cancers in the United States are squamous cell carcinomas (SCCs). However, worldwide, SCC is the most common form, accounting for 75% of bladder cancer in underdeveloped nations. In the United States, SCC is associated with persistent inflammation from long-term indwelling Foley catheters and bladder stones. In underdeveloped nations, SCC is associated with bladder infection by the parasite Schistosoma haematobium.

[0083] Adenocarcinomas account for less than 2% of primary bladder tumors. These tumors are observed most commonly in exstrophic bladders and respond poorly to radiation and chemotherapy. Radical cystectomy is the treatment of choice. Small cell carcinomas are aggressive tumors associated with a poor prognosis and are thought to arise from neuroendocrine stem cells. Carcinosarcomas are highly malignant tumors that contain both mesenchymal and epithelial elements.

[0084] For superficial (low grade) cancer, intravesical immunotherapy is available, such as Bacillus Calmette-Guerin (BCG) immunotherapy. This and other cures and therapies for cancers described herein may be used as a secondary treatment according to the invention. BCG immunotherapy may help to decrease the rate of recurrence and progression. It involves a live attenuated strain of Mycobacterium bovis. Some early studies purported that an immune response against BCG surface antigens cross-reacted with putative bladder tumor antigens, and this was proposed as the mechanism for the therapeutic effect of BCG; however, multiple subsequent studies refute this claim and demonstrate that BCG induces a nonspecific, cytokine- mediated immune response to foreign protein.

[0085] However, treatment with BCG has drawbacks. Because BCG is a live attenuated organism, it can cause an acute disseminated tuberculosis-like illness if it enters the bloodstream (BCG sepsis), possibly resulting in death. Therefore, the use of BCG is contraindicated in patients with gross hematuria. More mild side effects may include granulomatous cystitis or prostatitis with bladder contraction. Other drawbacks include the fact that this therapy is less effective in reducing the 5-year recurrence rate for low-grade and low-stage bladder cancer. [0086] Another treatment is interferon alpha or gamma. Interferon alpha or gamma has been used in the treatment of superficial bladder cancer, either as a single agent therapy or in combination with BCG. Its role has primarily been in post-BCG failure with early promising results. Although BCG with interferon has shown a 42% response with tolerable side effects after BCG failure, no evidence has indicated that re-treating with BCG with interferon is superior to re-treating with BCG alone.

[0087] Valrubicin has recently been approved as intravesical chemotherapy for bladder cancer that is refractory to BCG. In patients whose conditions do not respond to BCG, the overall response rate to valrubicin is approximately 20%, and some patients can delay time to cystectomy. Unfortunately, valrubicin is presently not commercially available. [0088] Other forms of adjuvant intravesical chemotherapy for superficial bladder cancer include intravesical triethylenethiophosphoramide (thiotepa [Thioplex]), mitomycin-C, doxorubicin, and epirubicin. Although these agents may increase the time to disease recurrence, no evidence indicates that these therapies prevent disease progression. Also, no evidence suggests that these adjuvant therapies are as effective as BCG.

[0089] For more advanced bladder cancers, such as those cancers involved in muscle- invasive disease (clinical stage T2 and greater), other pharmaceutical therapies include adjuvant and neoadjuvant chemotherapy. Neoadjuvant chemotherapy prior to either radical cystectomy or external beam radiotherapy is controversial as it is not clear whether it provides additional benefits to patients.

[0090] In one small series, the T4 tumors of 45% of affected patients responded to chemotherapy, making potentially curative cystectomy possible. Although no definite evidence of benefit exists, patients with P3-P4 or N+ bladder cancer in the United States are typically advised to receive adjuvant chemotherapy. Chemotherapeutic agents for metastatic disease include MVAC, which is the standard treatment of metastatic bladder cancer. MVAC has an objective response rate of 57-70%, a complete response rate of 15-20%, and a 2-year survival rate of 15-20%.

[0091] The combination of gemcitabine and cisplatin (GC) is a newer regimen and has been shown to be as efficacious as MVAC, but with less toxicity. GC is now considered a first- line treatment agent for bladder cancer.

[0092] Several novel compounds have shown activity against transitional cell bladder cancer and are now being tested in combination chemotherapy trials. Some of these promising agents are ifosfamide, paclitaxel, docetaxel, oxaliplatin and carboplatin. These may be used as secondary treatments.

[0093] In some embodiments, the compositions and methods of the present invention can be used to treat gliomas. As used herein, the term "glioma" encompasses, without limitation, gliomas, glioblastoma, Glioblastoma Multiforme (GBM), grade IV astrocytoma, grade IV glioma, high-grade astrocytoma, HGA, high-grade glioma, primary brain tumor, gliosarcoma, multifocal GBM, gliomatosis cerebri, intracranial tumor, tuberous sclerosis, neurofibromatosis type 1, neurofibromatosis type 2, Turcot syndrome, Li-Fraumeni syndrome, glioblastoma multiforme, brain tumors, malignant astrocytoma, de novo glioblastomas, secondary glioblastomas and primary glioblastoma.

[0094] GBM is an anaplastic, highly cellular tumor with poorly differentiated, round, or pleomorphic cells, occasional multinucleated cells, nuclear atypia, and anaplasia. Under the modified WHO classification, GBM differs from anaplastic astrocytomas (AA) by the presence of necrosis under the microscope. Variants of the tumor include gliosarcoma, multifocal GBM, or gliomatosis cerebri (in which the entire brain may be infiltrated with tumor cells). These variants, however, do not alter the prognosis of the tumor. Seldom do GBMs metastasize to the spinal cord or outside the nervous system.

[0095] The etiology of GBM is unknown. However, at least two genetic pathways have been delineated in its development: de novo (primary) glioblastomas and secondary glioblastomas. De novo glioblastomas are most common. De novo GBM develops in older patients and demonstrates a high rate of epidermal growth factor receptor (EGFR) overexpression, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutations, and pl6INK4A deletions. In contrast, secondary GBM develops in younger patients and develops from a malignant transformation of a previously diagnosed low-grade tumor. TP53 and retinoblastoma gene (RB) mutations are more common in the development of secondary glioblastomas.

[0096] Known treatments may be used as secondary treatments according to the invention. In GBM, surgery is typically performed yet cannot remove all of the tumor, since it is a highly infiltrating tumor. After surgery, radiation therapy remains the most effective adjuvant therapy for the treatment of patients with HGA/GBM. Radiotherapy prolongs the median survival by 14-36 weeks. Different methods of administering radiation therapy are available, including external beam radiation; stereotactic brachytherapy; stereotactic radiosurgery; and boron neutron capture therapy.

[0097] Chemotherapy may be used in combination with radiation therapy after surgery, although its effects are limited. Current recommendations include maximal possible surgical resection followed by concurrent radiation and chemotherapy with temozolomide.

[0098] A phase III randomized trial that included 240 patients compared surgery with implantation of polymer wafers with BCNU (Gliadel wafers) into the tumor bed demonstrated significant prolongation of survival compared with a placebo wafer. Both groups received radiation therapy. The median survival was 13.9 months in the group treated with Gliadel wafers and 11.6 months in the group treated with placebo.

[0099] For tumor recurrence, various conventional chemotherapeutic agents, including nitrosoureas such as BCNU and CCNU, and chemotherapies such as cisplatin, oxaliplatin, carboplatin, etoposide, are used. However their efficacy is limited.

[00100] In some embodiments, the methods of the present invention comprise administering a secondary therapeutic agent.

[00101] In some embodiments, the methods of the present invention comprise performing a secondary procedure. The term "secondary procedure" encompasses, without limitation, radiation treatment, surgery or a combination thereof. "Secondary procedure" also encompasses any procedure that can accompany administration of an HTR2A antagonist for treatment of any of the cancers described herein. As used herein, "perform" encompasses completing the procedure to the point that the procedure is, or within reasonable medical judgment should, be effective to treat the cancer, whether alone or in combination with an HTR2A antagonist. [00102] The HTR2A antagonist and the secondary treatment can be administered simultaneously or sequentially. For example, the HTR2A antagonist can be administered before or after the secondary treatment. As used herein, "sequentially" encompasses administering the HTR2A antagonist before or after administering or performing the secondary treatment. As used herein, "simultaneously" encompasses treatment regimens in which the start and end of both HTR2A antagonist and the secondary treatment occur at the same time. It also encompasses situations in which the HTR2A antagonist treatment and the secondary treatment overlap, without regard to whether they are started or completed at the same time or at different times. In other words, if the HTR2A antagonist and the secondary treatment are administered or performed at the same time at any point during the treatment regimen, they are considered to constitute simultaneous treatment as used herein. The invention contemplates administering the HTR2A antagonist early in therapy, and administering the secondary treatment later in therapy, optionally with an overlap, and optionally beginning both treatments simultaneously. [00103] The HTR2A antagonist and secondary therapeutic agent can be administered as part of a single composition or in separate compositions. The compositions can be in different types for formulations and be administered via different methods. For example, one can be formulated for intraperitoneal administration and the other can be formulated for subdermal implantation. The HTR2A antagonist can be administered before the secondary treatment, or the secondary treatment can be performed or administered before the HTR2A antagonist. The HTR2A antagonist and / or the secondary treatment can be administered along with any other suitable chemotherapeutic agent, according to the present invention. It has surprisingly been discovered that the HTR2A antagonist and the secondary treatment can act synergistically to more effectively treat cancer.

[00104] In some embodiments, the invention provides a combination therapy comprising administering 1/10 the indicated dosage of the secondary therapeutic agent in combination with an HTR2A antagonist, wherein the combination therapy inhibits cell proliferation of cancer cells approximately equal to use of the indicated dosage of the secondary therapeutic agent. As used herein, "combination therapy" refers to a therapeutic regimen involving administration of an HTR2A antagonist as well as administration of or performance of a secondary treatment, whether simultaneously or sequentially. As used herein, "indicated dosage" refers to the dosage level appropriate to treatment of cancer using the specified agent by itself, without a secondary treatment.

[00105] Use of HTR2A antagonists, including tetracyclic antidepressants such as mianserin, for treatment of cancer is different in surprising and unexpected ways over the known use of, for example, mianserin for treatment of depression. For example, treatment of cancer with tetracyclic antidepressants such as mianserin involves a different treatment duration, for example from about 1 month to about 1 year or more, or from about 1 month to about 3 months. Treatment of cancer with tetracyclic antidepressants such as mianserin involves different dosages, for example at least about 100 mg/day for mianserin. It also involves a different patient population: those with cancer, regardless of the presence of other disorders or conditions, rather than those with depression. It also can involve treatment of the cancer by administering a composition of the invention without diagnosing depression in the patient. Cancer patients who do not exhibit clinical symptoms of depression, as measured by, for example, Research Diagnostic Criteria or the Diagnostic and Statistical Manual of Mental Disorders ("DSM"), latest version, can be treated according to the present invention. The compositions and methods of the present invention can use a wide range of tetracyclic antidepressants, for example amoxapine, maprotiline, mirtazapine, and/or trazodone; and they can be used to treat several forms of cancer, such as urologic cancer, glioma and/or hormone-refractory prostate cancer. [00106] Suitable routes of administration include, without limitation, oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient's body, such as for example through the urethra (for transurethral administration).

[00107] It will be appreciated that treatment of the diseases described herein according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). Thus, treatment of malignancies using HTR2A antagonists may be combined with, for example, radiation therapy, antibody therapy and/or chemotherapy. Optionally, treatment with one or more of a secondary treatment or treatments, sequentially or simultaneously, is also contemplated.

[00108] For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[00109] For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[001 10] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[00111] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-tit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[00112] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[00113] For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[00114] The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[00115] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[00116] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[00117] The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[00118] Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[00119] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[00120] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics",

Ch. 1 p.l).

[00121] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. [00122] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Typical dosages and routes of administration for several agents are presented in the following table. This information is for illustrative purposes only and is not intended to be limiting.

Kits

[00123] In another aspect, the invention provides kits comprising a therapeutically effective amount HTR2A antagonist for the treatment of cancer, and a container, optionally with labeling. The kit can be an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. In some embodiments, the kit may also comprise a secondary therapeutic agent.

[00124] The container can be any suitable container capable of holding and dispensing the dosage form and which will not significantly interact with the composition. The pack may, for example, comprise metal or plastic foil, such as a blister pack, or it can be any other suitable dispenser device.

[00125] In some embodiments, a label is associated with the container. For example, the printed labeling may provide instructions for administering any of the compositions, including administering an HTR2A antagonist and a secondary therapeutic agent; using any of the kits; or performing any other method herein described. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. The labeling instructions will be consistent with the methods of treatment described herein. The labeling may be associated with the container by any means that maintain a physical proximity of the two. By way of non-limiting example, they may both be contained in a packaging material such as a box or plastic shrink wrap or may be associated with the instructions being bonded to the container such as with glue that does not obscure the labeling instructions or other bonding or holding means.

Diagnostic methods

[00126] In another aspect, the invention provides methods of diagnosing whether a form of cancer is responsive to an HTR2A antagonist. In some embodiments, the methods comprise isolating tissue, e.g. by biopsy, measuring HTR2A activity in the tissue, and identifying tissue having elevated HTR2A activity as potentially responsive to an HTR2A antagonist for treatment of cancer, such as breast cancer, urologic cancer, hormone-refractory prostate cancer and a glioma. In some embodiments, measuring HTR2A activity comprises measuring expression of HTR2A protein or measuring ability to bind serotonin in cells of a patient. In some embodiments, measuring HTR2A expression is carried out as described below in Example 1. [00127] If the cancerous tissue is identified as potentially responsive to an HTR2A antagonist, therapy according to the invention may be prescribed and administered. Progress of therapy may be monitored by these methods as well.

Assay methods

[00128] In still another aspect, the invention provides methods of assaying for anti-cancer or anti-proliferative activity of a compound of interest. In some embodiments, the methods comprise measuring HTR2A activity in a cancer cell line, contacting the cancer cell line with a compound of interest; and determining if the compound reduces HTR2A activity in the cell line. The method may comprise determining if the compound reduces proliferation of the cancer cell line relative to a control. The methods may comprise contacting the cancer cell line with a secondary therapeutic agent, and determining if the secondary therapeutic agent further reduces HTR2A activity in the cell line, and/or determining whether the secondary therapeutic agents further reduces proliferation of the cancer cell line.

[00129] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. EXAMPLES

[00130] EXAMPLE 1: Expression of the 5-hydroxytryptamine [serotonin] receptor

2A (HTR2A) in human breast cancer cell lines

[00131] Two human breast cancer cell lines MCF7 cells (Adenocarcinoma, Breast, ER positive, HTB-22, ATCC) or MDA-MB-468 cells (Adenocarcinoma, Breast, ER negative, HTB- 132, ATCC) were used to examine the expression of the HTR2A protein. The cells were lysed and the levels of HTR2A expression were determined by immunoblotting with an antibody against HTR2A. Blots were reprobed with an anti-tub ulin antibody, and the expression levels of HTR2A were normalized to tubulin protein levels. [00132] Cell treatments and lysis:

[00133] MCF7 cells and MDA-MB-468 cells were grown in T-75 flasks according to the

ATCC guidelines. Medium was removed, cells were washed with ice-cold Dulbecco's PBS (Biological Industries, Israel, Cat. No. 02-023- IA), and 500 μl of lysis buffer (50 mM Tris pH 7.4, 1% NP-40, 2 mM EDTA, 100 mM NaCl) containing complete protease inhibitor cocktail (Roche, 1-873-580-001), were added to each flask. Flasks were incubated on ice for 30 minutes and tapped until cells dissociation occurred. Lysates were transferred to 1.5ml tube, centrifuged at 4⁰C for 10 minutes at 14,000 rpm, and sup was transferred to new tubes. [00134] Immunoblot Analysis:

[00135] Lysate samples were separated on 4-12% Bis-Tris gels (Invitrogen) in NuPAGE

MOPS nrnning buffer (Invitrogen, NPOOOl). Proteins were transferred to nitrocellulose membranes using NuPAGE transfer buffer (Invitrogen, NP0006). After transfer, blots were stained with Ponceau S solution (Sigma, Cat. No. P-7170), and washed twice with TBS-T 0.1% (TBS with 0.1% Tween-20). Blocking was earned out at RT for lhr with 5% nonfat dry milk (BD, Difco skim milk, 232100, Lot.4184250) in TBS-T 0.1%. Anti-HTR2A, monoclonal ab. (BD Pharmingen, Cat # 556326 Lot. 47700) was added at 1:500 in blocking buffer and incubated overnight at 4⁰C.

[00136] The blot was washed three times in TBS-T 0.1%, and secondary Ab, peroxidase- conjugated goat-anti-mouse (Jackson, 115-035-146, Lot. 63343) 1:50,000 was added in blocking solution for 2 hrs at RT. The blot was washed three times in TBS-T 0.1% and SuperSignal West Pico Chemiluminiscent (Pierce, Cat. No. 34080) was used for detection of HRP. Equal volumes of each solution were mixed; the blot was immersed in the solution for 5 min and exposed to film. [00137] For reprobing with anti-Tubulin Ab, the blot was stripped in Restore buffer

(Pierce, cat#21059, lot#HD103652) for 30 min at RT. The blot was washed three times with TBS-T-0.1% and re-blocked with 5% dry skim milk (Difco, 232100, Lot. 4184250) in TBS- Tween 20, 0.1%, 1 hrs at RT followed by incubation with 1:30,000 anti-Tubulin Ab (mouse monoclonal Ab, kindly provided by Yinon Ben-Neria [Haddasah Medical School, The Hebrew University of Jerusalem]) for overnight at 4⁰C. Blots were washed as above, and secondary Ab, goat-anti-mouse (Jackson ImmunoResearch, 1 15-035-146) was added at 1:25,000 in blocking solution, for lhr at RT. Blots were washed again, and HRP detection was earned out with SuperSignal West Pico Chemilummiscent as described above. Autoradiograms were scanned and levels of HTR2A protein were quantified by densitometry using ImageJ 1.36b software, and normalized to levels of Tubulin expression. [00138] Results:

[00139] The results of the immunoblotting described above are shown in Figure 1. As can be seen in Figure 1, both breast cancer cell lines express the HTR2A protein, though at different levels. MCF7 expresses 2.6 times more of the HTR2A protein than the MDA-MB-468 cell line.

[00140] EXAMPLE 2: Effect of Mianserin on cell proliferation of human cancer cell lines

[00141] The effect of Mianserin on proliferation of MCF7 cells (Adenocarcinoma, Breast,

ER positive, HTB-22, ATCC), MDA-MB-468 cells (Adenocarcinoma, Breast, ER negative, HTB-132, ATCC), U87-MG cells (Glioblastoma, Brain, HTB- 14, ATCC), T98G cells (Glioblastoma, Brain, CRL-1690, ATCC) or SCaBER cells (Bladder, HTB-3, ATCC) was tested using BrdU incorporation assay. Surprisingly it was found that Mianserin was effective against bladder cancer cells and glioma (glioblastoma) cancer cells, in addition to being effective against breast cancer cells.

[00142] Description of BrdU proliferation assay:

[00143] MCF7, MDA-MB-468, U87-MG, T98G or SCaBER cells were grown according to the ATCC guidelines. Cells were seeded in 96-well microtiter plates at a concentration of 8,000 cells/well (MCF7, MDA-MB-468, U87-MG) or 5,000 cells/well (T98G, SCaBER) in a final volume of 200 μl medium containing 10% FBS (Fetal bovine serum, Heat Inactivated, Biological Industries, Cat.No.04-121 -IA). On the next day, cells were rinsed and supplemented with 200 μl of medium containing 0.1 % FBS for additional 24 hrs. After serum starvation, cells were treated with different concentrations of Mianserin (Sigma-Alrich, Israel, Cat # M 2525, lot # 036Kl 217, dissolved in double distilled water) and/or with Carboplatin (Sigma-Alrich, Israel, C2538, Lot.066kl724, CAS # 41575-94-4, dissolved in double distilled water) and/or with Tamoxifen (Sigma-Alrich, Israel, Cat # T 5648, lot # 046Kl 569, CAS # 10540-29-1, dissolved in DMSO), for 72 hrs. For BrdU incoiporation assay, BrdU was added at a final concentration of 10 μM for 2 hrs. BrdU ELISA assay was performed according to the manufacturer instructions (Cell proliferation ELISA, Roche, Cat. No.l 1 647 229 001). [00144] Results:

[00145] The results of the proliferation assays described above are shown in Figure 2. As can be seen in Figures 2A, 2B, 2C, 2D and 2E, Mianserin inhibits the proliferation of MCF7, MDA-MB-468, T98G, U87-MG and SCaBER cells, respectively, as measured by BrdU incoiporation. The results, which are depicted as relative growth compared to untreated cells (which defined as 100), indicate a dose dependent inhibition of proliferation by HTR2A antagonist in all five cell lines. Figure 2F shows the affect of Carboplatin on proliferation of MCF7 cells with or without Mianserin at different concentrations. Results indicate a synergistic effect for the two drugs. For example, 3 μM of Carboplatin together with 1 OμM of Mianserin show a similar effect to that of 30μM Carboplatin. Figure 2G shows the affect of Tamoxifen on proliferation of MCF7 cells with or without Mianserin at different concentrations. Results indicate a synergistic effect for the two drugs. For example, 1 μM of Tamoxifen together with 1 OμM of Mianserin show a similar effect to that of 10 μM Tamoxifen.

[00146] EXAMPLE 3: Effect of Mianserin and Tamoxifen on Induced Tumors in Mice

[00147] Materials and Methods [00148] Test substances

[00149] Mianserin from Sigma (Ref M2525, Saint Quentin Fallavier, France). Substance characteristics are described in the following table:

[00150] Mianserin (MIA) as powder was stored at 2-8°C as indicated by Sigma. A stock solution of Mianserin at 15 mg/ml was prepared under laminar flow conditions by dissolution in 0.9% saline solution. Further appropriate dilutions were prepared freshly in 0.9% saline solution. [00151] Tamoxifen (TAM) free-base pellets (Ref SE-361, 2.5 mg/pellet, 60-day release,

Innovative Research of America, Saratosa, Floride, USA) were used alone or in combination with the test substance. Tamoxifen pellets were stored at room temperature following the instructions provided by the supplier. Pellets were SC implanted at the base of neck of mice with a 10-G trochar supplied from Innovative Research of America (catalog number: MP-182). [00152] One treatment dose of Mianserin was 25mg/kg based on the results of a tolerance study. The administration route of Mianserin is intraperitoneally (IP). Administration volume was 10 ml/kg/injection (e.g. 200 μl/mouse weighing 20 g) adjusted to the most recent individual body weight of mice.

[00153] One treatment dose of Tamoxifen was a 2.5 mg-Tamoxifen, administered via an implanted pellet with a 60-day release. [00154] Animals

[00155] Fifty six healthy female CB- 17 Balb/c nu/nu mice, 5-6 week-old, weighing 18-

2Og were obtained from Charles River (L'Arbresle, France). Nine were used for the single tolerance study, 3 for the combined tolerance study, and 44 for antitumor activity study. Animals were observed for 7 days in a specific-pathogen-free (SPF) animal care unit before use. The animal care unit is authorized by the French ministries of Agriculture and Research (Agreement No A21231011). Animal experiments were performed according to ethical guidelines of animal experimentation (Principe d'ethique de ^experimentation animale, Directive n°86/609 CEE du 24 Nov. 1986, Decret n°87/848 du 19 Oct. 1987, Arrete d'Application du 19 Avril 1988) and the British guidelines for the welfare of animals in experimental neoplasia (WORKMAN P. et al, UKCCCR guideline, Br. J. Cancer, 1998, 77: 1- 10.). All procedures with animals will be submitted to the Animal Care and Use Committee of Pharmacy and Medicine University (Dijon). Animals were maintained in rooms under controlled conditions of temperature (23 ± 2°C), humidity (45 ± 10%), photoperiod (12h light/ 12h dark) and air exchange. Animals were housed in polycarbonate cages (Techniplast, Limonest, France) that are equipped to provide food and water. Animal food was purchased from SERLAB. Water was supplemented with 2.5 μg/ml estradiol and also provided ad libitum from water bottles equipped with rubber stoppers and sipper tubes. [00156] Tumor cell line

[00157] MCF-7 tumor cell line was obtained from Jules Bordet Institute (Brussels,

Belgium).

[00158] MCF-7 cell line was established from the pleural effusion of a 69-year old

Caucasian female patient with a metastatic mammary carcinoma (after radio- and hormone therapies) (BURK K.H. et al., Cancer Res., 1978, 38: 2508-2513.).

[00159] Tumor cells were grown as adherent monolayer at 37°C in a humidified atmosphere (5% CO₂, 95% air). The culture medium was RPMI 1640 including 2 mM L-glutamine (Ref. BE12-702F, Lonza, Verviers) supplemented with 10% fetal bovine serum (Ref. DE 14-80 IE, Lonza, Verviers). The cells are adherent to plastic flasks. For experimental use, adherent tumor cells were detached from the culture flask by 5-minute treatment with trypsin-versene (Ref. BEl 7-16 IE, Lonza, Verviers), diluted in Hanks' medium, without calcium or magnesium (Ref. BE10-543F, Lonza, Verviers) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.

[00160] Experimental Design and Treatments

[00161] Tolerance study of Mianserin in healthy Balb/c Nude mice

[00162] Before the start of treatments, 9 healthy female Balb/c Nude mice were randomized based on body weight into 3 groups of 3 animals. The mean body weight of each group was comparable and not statistically different from the other groups (analysis of variance). [00163] The mice from group 1 received daily repeated IP injections of Mianserin, at

150 mg/kg/injection for 14 consecutive days (schedule named QlDxl4). The mice from group 2 receive daily repeated IP injections of Mianserin, at 100 mg/kg/injection for 14 consecutive days (schedule named Q IDx 14). The mice from group 3 receive daily repeated IP injections of Mianserin, at 50 mg/kg/injection for 14 consecutive days (schedule named QlDxl4). Mouse survival 7 days after injection was the end point of experiment. [00164] The treatment schedule is summarized in the table below:

[00165] Tolerance of Mianserin combined to Tamoxifen in Balb/c Nude mice

[00166] Three healthy female Balb/c Nude mice with comparable body weight were used for the combined tolerance study. The mice from group 1 receive daily repeated IP injections of

Mianserin, at dose #1 (based on the results of the first tolerance study) for 14 consecutive days

(schedule named QlDxH), combined with 2.5 mg-tamoxifen implanted pellet (60-day release).

Mouse survival 7 days after injection was the end point of experiment.

[00167] The treatment schedule is summarized in the table below:

[00168] Antitumor activity study of Mianserin combined to Tamoxifen in Balb/c Nude mice SC xenografted with MCF-7 tumor cells

[00169] MCF-7 tumor cells were amplified in vitro in complete culture medium. Cells were collected by trypsinisation, centrifuged at 1,000 rpm for 5 minutes, washed using

RPMI 1640 medium, and resuspended to reach a concentration of 5 x 10⁷ cells/ml of RPMI 1640 medium. Tumors were induced SC by injecting 10⁷ cells in 200 μl of RPMI 1640 into the right flank of 44 Balb/c Nude mice, 24 to 48 hours after a whole body irradiation with a γ-source

(1.8 Gy, ⁶⁰Co, INRA BRETENIERES, Dijon, France). Tumor induction is considered as day 0

(DO).

[00170] Treatment started when the tumors reach a mean volume of 100-200 mm³ (DO).

Before the start of treatments, 40 mice out of 44 were randomized according to their individual tumor volume into 4 groups of 10 mice. The mean tumor volume of each group was comparable and not statistically different from the others (analysis of variance).

[00171] The treatment schedule was as follows: Group 1: 10 mice received daily repeated

IP injections of vehicle for 14 consecutive days (schedule named QlDxH). Group 2: 10 mice received daily repeated IP injections of Mianserin at dose #1 (based on the results of tolerance studies) for 14 consecutive days (schedule named QlDxl4). Group 3: 10 mice received daily repeated IP injections of Mianserin at dose #1 for 14 consecutive days (schedule named Ql DxI 4), combined with 2.5 mg-tamoxifen implanted pellet (60-day release). Group 4: 10 mice were implanted with one 2.5 mg-tamoxifen implanted pellet (60-days release). Surviving mice in treated group were sacrificed when control mice reach two times the mean survival time. [00172] The treatment schedule is summarized in the table below:

[00173] Isoflurane /Errane^® (TEM, Bordeaux, France) was used to anaesthetize the animals before SC injections of tumor cells, pellet implantation, and at sacrifice. Mortality and behaviour was recorded every day. Mouse body weight and the tumor volume was monitored and recorded twice a week. During the course of the experiment, animals were killed by cervical dislocation while under isoflurane anaesthesia if any of the following occurred: signs of suffering (cachexia, weakening, difficulty moving or eating); compound toxicity (hunching, convulsions); tumor ulcerating and remaining open; tumor growing up to 2000 mm³; or 15% body weight loss over a period of 3 consecutive days or 20% body weight loss for 1 day. [00174] An autopsy (macroscopic examination) was performed on all sacrificed

(scheduled) mice in the study, and, if feasible, on all moribund/found dead mice (SOP TEC- 090/001). Autopsy observations were recorded. [00175] Data Management:

[00176] Tumor size was measured twice a week with a calliper, and tumor volume (in mm³) was estimated by the formula (4):

_ , length x width²

Tumour volume = —

2

[00177] Treatment efficacy was assessed in terms of the effects of mianserin or mianserin combined with tamoxifen on the tumor volumes relative to vehicle mice:

[00178] Tumor growth curves of vehicle and treated groups using the mean tumor volumes (MTV) was drawn and provided along with growth curves showing data for individual mice in each group.

[00179] Tumor doubling time (DT), defined as the period required to reach a MTV of

200% during the exponential tumor growth phase was calculated. The calculation of DT was performed using GraphPad^® Prism software (San Diego, USA).

[00180] Tumor growth inhibition (T/C%) defined as the ratio of the median tumor volume of treated versus vehicle-treated groups was calculated as following:

T/C (%) = Median TV of treated group at D_{x χl QQ} Median TV of vehicle - treated group at D_x

[00181] The optimal value is a minimal T/C% ratio reflecting a maximal tumor growth inhibition. The effective criterion for the T/C% ratio according to NCI standards is < 42% (5). [00182] Relative tumor volume (RTV) curves of vehicle and treated groups were drawn, the RTV being calculated using the following formula:

RTV = ^{TV at D}«

TV at D_R

[00183] Where D_x= Day of tumor volume monitoring and D_R=Day of randomization

[00184] Volume V and Time to reach V was calculated and provided. Volume V is defined as a target volume deduced from experimental data and chosen in exponential phase of tumor growth. Volume V will be chosen as the same for all mice from all groups, the time to reach this Volume V will be determined from experimental data. [00185] All statistical analyses were performed using StatView'^® software (Abacus

Concept, Berkeley, USA). The log-Rank (Kaplan-Meier) was used to compare the survival curves. Statistical analysis of MBWC, volume V and time to reach V, and DT was performed using the Bonferroni/Dunn test (ANOVA comparison). All groups were compared with each other. A p value < 0.05 will be considered as significant. [00186] Results

[00187] Tamoxifen induced in this model a significant antitumor activity (T/C%= 30% at

D56) and the combination of MIA+TAM exhibited superior results (T/C%= 23% at D56) (Table 1 and Figure 3). MIA alone induced a slight antitumor activity at the early phase of treatment (Figure 3). By itself, MIA did not have significant antitumor activity at Day 56, although it did inhibit early growth (see Day 28). Also, there was an unexpected synergistic effect with the primary and secondary treatments combined. It has been noticed that treatment response was faster when mice were treated with TAM+MIA. Indeed, T/C% of the TAM+MIA treated group is lower than 50% as early as D25 (4 days after first treatment, Table 1), while the TAM only treated group did not reach a T/C% below 50% until D39. The tumor growth delay was significantly increased, with the MIA / TAM combination being surprisingly more effective than TAM alone at delaying tumor growth to 400 mm³ and 600 mm³ (Table T). The relative tumor volume curves showed similar conclusion with an early phase where TAM+MIA is slightly better than TAM alone. The tumor volume at the day of randomisation did not seem to impact the treatment response.

[00188] The results are summarized in Tables 1-3, below. Data is presented for mice that survived and also achieved the criteria evaluated. For example, five mice in the MIA arm survived and also achieved a tumor volume of 400 mm³. Several mice died during the course of the study. In the vehicle arm, two mice were sacrificed on day 60 after their tumors reached the upper limit of tumor volume allowed (i.e., 2000 mm³). In the MIA arm, four mice were found dead on day 25. In the TAM+MIA arm, three mice died, one each on days 25, 27 and 49. In the TAM arm, no mice died during the course of the study. 95798-258468

002006

[00189] EXAMPLE 4: Effect of other HTR2A antagonists on cell proliferation of human cancer cell lines

[00190] Example 4A: Amoxapine

[00191] The protocol described in Example 2 is performed using amoxapine instead of mianserin. Amoxapine is effective at inhibiting proliferation of MCF7, MDA-MB-468, T98G,

U87-MG and SCaBER cells.

[00192] Example 4B: Maprotiline

[00193] The protocol described in Example 2 is performed using maprotiline instead of mianserin. Maprotiline is effective at inhibiting proliferation of MCF7, MDA-MB-468, T98G,

U87-MG and SCaBER cells.

[00194] Example 4C: Mirtazapine

[00195] The protocol described in Example 2 is performed using mirtazapine instead of mianserin. Mirtazapine is effective at inhibiting proliferation of MCF7, MDA-MB-468, T98G,

U87-MG and SCaBER cells.

[00196] Example 4D: Trazodone

[00197] The protocol described in Example 2 is performed using trazodone instead of mianserin. Trazodone is effective at inhibiting proliferation of MCF7, MDA-MB-468, T98G,

U87-MG and SCaBER cells.

[00198] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[00199] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims

B2008/002006CLAIMS

1. A method for treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an HTR2A antagonist, thereby treating the cancer.

2. The method of claim 1 , wherein the HTR2A antagonist is a tetracyclic antidepressant.

3. The method of claim 2, wherein the tetracyclic antidepressant comprises mianserin.

4. The method of claim 3, wherein the mianserin is administered at a dosage of at least about 100 mg daily.

5. The method of claim 2, wherein the tetracyclic antidepressant is selected from the group consisting of amoxapine, maprotiline, mirtazapine, trazodone, and combinations thereof.

6. The method of claim 2, wherein the cancer is selected from the group consisting of breast cancer, urologic cancer, glioma and hormone-refractory prostate cancer.

7. The method of claim 2, wherein the cancer is urologic cancer

8. The method of claim 2, wherein the cancer is glioma.

9. The method of claim 2, wherein the cancer is hormone-refractory prostate cancer.

10. The method of claim 2, wherein the cancer is breast cancer.

11. The method of claim 2, further comprising administering a secondary therapeutic agent.

12. The method of claim 11, wherein the secondary therapeutic agent is selected from the group consisting of retinoids, cyclooxygenasc (COX) inhibitors, estrogen receptor modulators, aromatase inhibitors, platin-based chemotherapeutic agents, progesterone- like drugs, anti-hormone agents, trastuzumab, MVAC, taxol, taxotere, gemcitabine, GC, temozolomide, BCNU, CCNU, etoposide, leuprolide, goserelin and combinations thereof.

13. The method of claim 12, wherein the estrogen receptor modulator is selected from the group consisting of tamoxifen, toremifene, fulvestrant, and combinations thereof.

14. The method of claim 12, wherein the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole, exemestane, and combinations thereof.

15. The method of claim 12, wherein the platin-based chemotherapeutic agent is selected from the group consisting of carboplatin, oxaliplatin, cisplatin, and combinations thereof.

16. The method of claim 12, wherein the progesterone-like drug is megestrol acetate.

17. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is an anti-hormone agent.

18. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is tamoxifen.

19. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is toremifene.

20. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is fulvestrant.

21. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is letrozole.

22. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is anastrozole.

23. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is platin-based chemotherapeutic agent.

24. The method of claim 11 , wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is carboplatin.

25. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is oxaliplatin.

26. The method of claim 11, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is cisplatin.

27. The method of claim 11, wherein the tetracyclic antidepressant and the secondary therapeutic agent are administered sequentially.

28. The method of claim 11, wherein the tetracyclic antidepressant and the secondary therapeutic agent are administered simultaneously.

29. The method of any of claims 1-28, comprising administering the pharmaceutical composition without diagnosing depression in the patient.

30. The method of any of claims 1-28, wherein the patient does not exhibit clinical symptoms of depression.

31. The method of any of claims 1-28, comprising administering the pharmaceutical composition daily for a period of about 1 month to about 3 months.

32. The method of any of claims 1-28, comprising administering the pharmaceutical composition daily for a period of about 1 month to about 1 year.

33. The method of claim 1, wherein the pharmaceutical composition is administered orally, rectally, transmucosally, transnasally, intestinally, parenteral Iy, intramuscularly, subcutaneously, intramedullarily, intrathecally, intraventricularly, intravenously, intraperitoneally, intranasally, intraocularly, or transurethrally.

34. The method of claim 2, further comprising performing a secondary procedure.

35. The method of claim 34, wherein the secondary procedure comprises surgery.

36. The method of claim 34, wherein the secondary procedure comprises radiation treatment.

37. The method of claim 34, wherein the secondary procedure is performed prior to administering the therapeutically effective amount of the pharmaceutical composition.

38. The method of claim 34, wherein the secondary procedure is performed after administering the therapeutically effective amount of the pharmaceutical composition.

39. A pharmaceutical composition for combination therapy comprising a tetracyclic antidepressant and a secondary therapeutic agent, wherein the secondary therapeutic agent has a first effective dosage for treating cancer when administered alone, and the dosage in the pharmaceutical composition is lower than the first effective dosage of the secondary therapeutic agent, the combination therapy achieving at least substantially the same efficacy in inhibiting cancer cell growth as the secondary therapeutic agent achieves when administered alone in the first effective dosage.

40. A pharmaceutical composition comprising an HTR2A antagonist in a therapeutically effective amount to treat cancer.

41. The composition of claim 40, wherein the HTR2A antagonist is a tetracyclic antidepressant.

42. The composition of claim 41, wherein the cancer is selected from the group consisting of breast cancer, urologic cancer, glioma and hormone-refractory prostate cancer.

43. The composition of claim 41, wherein the tetracyclic antidepressant is selected from the group consisting of amoxapine, maprotiline, mirtazapine, trazodone, and combinations thereof.

44. The composition of claim 41, wherein the tetracyclic antidepressant is mianserin and the mianserin is administered in a dosage of at least about 100 mg daily.

45. The composition of claim 41, further comprising a secondary therapeutic agent.

46. The composition of claim 45, wherein the secondary therapeutic agent is selected from the group consisting of retinoids, cyclooxygenase (COX) inhibitors, estrogen receptor modulators, aromatase inhibitors, anti-hormone agents, platin-based chemotherapeutic agents, trastuzumab, MVAC, taxol, taxotere, gemcitabine, GC, temozolomide, BCNU, CCNU, etoposide, progesterone-like drugs, goserelin and combinations thereof.

47. The method of claim 46, wherein the estrogen receptor modulator is selected from the group consisting of tamoxifen, toremifene, fulvestrant, and combinations thereof.

48. The method of claim 46, wherein the aromatase inhibitor is selected from the group consisting of anastrozole, letrozole, exemestane, and combinations thereof.

49. The method of claim 46, wherein the platin-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, and combinations thereof.

50. The method of claim 46, wherein the progesterone-like drug is megestrol acetate.

51. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is an anti-hormone agent.

52. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is tamoxifen. T/IB2008/002006

53. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is toremifene.

54. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is fulvestrant.

55. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is letrozole.

56. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is anastrozole.

57. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is platin-based chemotherapeutic agent.

58. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is carboplatin.

59. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is oxaliplatin.

60. The composition of claim 45, wherein the tetracyclic antidepressant is mianserin and the secondary therapeutic agent is cisplatin.

61. A kit comprising a pharmaceutical composition comprising a therapeutically effective amount of a tetracyclic antidepressant, a container, and a label associated with the container, wherein the label contains directions for administration of the pharmaceutical composition for treatment of cancer.

62. The kit of claim 61, wherein the container is a blister pack.

63. The kit of claim 61, wherein the tetracyclic antidepressant is mianserin. T/IB2008/002006

64. The kit of claim 61, comprising a pharmaceutical composition comprising a secondary therapeutic agent.

65. The kit of claim 64, wherein the label provides directions for administering the tetracyclic antidepressant and secondary therapeutic agent for treatment of cancer.

66. A method of diagnosing cancer that is responsive to an HTR2A antagonist, comprising: measuring HTR2A activity in a tissue, and identifying tissue having elevated HTR2A activity as potentially cancerous.

67. The method of claim 66, wherein measuring HTR2A activity comprises measuring expression of HTR2A protein or measuring ability to bind serotonin in cells of a patient.

68. A method of assaying activity of a compound of interest, comprising: measuring HTR2A activity in a cancer cell line; contacting the cancer cell line with a compound of interest; and determining if the compound reduces HTR2A activity in the cancer cell line.

69. The method of claim 68, further comprising the steps of: contacting the cancer cell line with a secondary therapeutic agent, and determining if the secondary therapeutic agent further reduces HTR2A activity in the cell line.

DC2/960840