DETAILED DESCRIPTION OF THE INVENTION

[0056] We have now demonstrated that the mechanism by which hypoxia and hyponitroxia have impact on cellular phenotypes is not mediated solely due to a lack of oxygen, but rather also from a deficiency in nitric oxide mimetic activity. Further, we have now demonstrated that administration of a low dose of a nitric oxide mimetic is sufficient to increase, restore or maintain levels of nitric oxide mimetic activity of cells so that a malignant cell phenotype is inhibited or prevented. This inhibition and prevention occurs even when the cells are in a hypoxic environment and/or when combined with inhibition of endogenous nitric oxide production. Nitric oxide mimetics are effective under normoxic conditions as well as hypoxic conditions. Administration of very low doses of nitric oxide mimetics, even under conditions of markedly reduced levels of oxygen (1% O ₂ ), was able to prevent the generation of a malignant cell phenotype and inhibit a malignant cell phenotype of cells.

[0057] Accordingly, the present invention relates to the use of nitric oxide mimetic (e.g., low dose) therapy in inhibiting and preventing a malignant cell phenotype of cells. The methods and formulations of the present invention provide new therapeutic approaches for the treatment and prevention of cancer in animals. For purposes of the present invention, by “treatment” or “treating” it is meant to encompass all means for controlling cancer by reducing growth of cells exhibiting a malignant cell phenotype and improving response to antimalignant therapeutic modalities. Thus, by “treatment” or “treating” it is meant to inhibit the survival and/or growth of cells exhibiting a malignant cell phenotype, prevent the survival and/or growth of cells exhibiting a malignant cell phenotype, decrease the invasiveness of cells exhibiting a malignant cell phenotype, decrease the progression of cells exhibiting a malignant cell phenotype, decrease the metastases of cells exhibiting a malignant cell phenotype, increase the regression of cells exhibiting a malignant cell phenotype, and/or facilitate the killing of cells exhibiting a malignant cell phenotype. “Treatment” or “treating” is also meant to encompass maintenance of cells exhibiting a malignant cell phenotype in a dormant (or quiescent) state at their primary site as well as secondary sites. Further, by “treating or “treatment” it is meant to increase the efficacy as well as prevent or decrease resistance to antimalignant therapeutic modalities. By “antimalignant therapeutic modalities” it is meant to include, but is not limited to, radiation therapies, thermal therapies, immunotherapies, hormone therapies, single agent chemotherapies, combination chemotherapies, chemo-irradiation therapies, adjuvant therapies, neo-adjuvant therapies, palliative therapies, and other therapies used by those of skill in the art in the treatment of cancer and other malignancies. “Treating” or “treatment” is also meant to encompass prolonged cancer remission, prevention of recurrence, decrease of cancer markers, reduction in cancer volume, reduction of pain, discomfort, and disability (morbidity), increase in quality of life associated with antimalignant therapeutic modalities, a decrease in mucositis, and a reduction in the need for anti-emetic agents and narcotic pain killers. By “increasing the efficacy”, it is meant to include an increase in potency and/or activity of the antimalignant therapeutic modality and/or a decrease in the development of resistance to the antimalignant therapeutic modality, and/or an increase in sensitivity of the malignant cells and/or tumor to the antimalignant therapeutic modality.

[0058] The present invention also relates to methods of monitoring and/or diagnosing malignant cell phenotypes in an animal via measurement of tumor selective markers in an animal in the presence of NO mimetic (e.g. low dose) therapy. Exemplary tumor markers useful in the monitoring and diagnosing of tumor progression and metastases include, but are not limited to, prostate specific antigen (PSA) for prostate cancer, carcinoembryonic antigen (CEA) and polypeptides such as gastrin and glucagon for gastrointestinal cancer, α-fetoprotein (AFP) and βhCG for testicular cancer, α-fetoprotein (AFP), human chorionic gonadotrophin (HCG) and lactate hydroginase (LDH) in germ cell cancers, HCG in choriocarcinoma, serum AFP in hepatocellular carcinoma, neuron-specific enolase (NSE) in small-cell lung cancer, paraprotein levels and B2-microglobulin which may be of prognostic value in myeloma, 5-hydroxyindole acetic acid (5HIAA) urine levels in carcinoid tumors, squamous cell carcinoma antigen (SCC) and cytokeratin fragments for squamous cell carcinoma, SCC and CA 125 for prognostic information in cervical carcinoma, CA19-9 and CA72-4 for gastric cancer, CD 24 for various cancers, including p-selectin ligand in non-small cell lung cancer, NFκB for prostate cancer prognosis, serum tumor marker CA125 for ovarian cancer, CDKN2A deletion as a diagnostic marker for malignant mesothelioma, dysadherin overexpression in pancreatic ductal adenocarcinoma, SMAD7 as a prognostic marker in patients with colorectal cancer, TGFβ1 as a potential prognostic marker for breast cancer patients with advanced disease, neuroendocrine and cytokeratin serum markers as prognostic determinants of small cell lung cancer, ErbB2 and bone sialoprotein as markers for metastatic osteosarcoma cells, cyclin E and CA15-3 for breast cancer and the cell surface receptors for estrogen and Her-2 for breast cancer. Additional markers which can be monitored for diagnostic purposes include, but are not limited to, calcitonin and calcitonin-related peptide for diagnosis and screening of medullary thyroid carcinoma, Protein Regulated by OXYgen-1 (PROXY-1), also known as NDRG-1, plasminogen activator inhibitor (PAI-1), urokinase-type plasminogen activator receptor (uPAR) and vascular endothelial growth factor (VEGF). Further, as will be understood by those of skill in the art upon reading this disclosure, additional tumor markers to those exemplified herein can also be monitored in the present invention. In a preferred embodiment, the tumor marker is detectable in a biological fluid such a serum, plasma or urine. No change, a decrease or deceleration in the increase of the level of one or more of these markers in an animal following administration of a low dose nitric oxide mimetic as compared to the level of the marker in the animal prior to administration of the low dose nitric oxide mimetic is indicative of a malignant cell phenotype in the animal.

[0059] For purposes of the present invention by the term “low dose” it is meant an amount of nitric oxide mimetic which is capable of increasing, restoring or maintaining a level of nitric oxide mimetic activity to cells, tumors and/or diseases which inhibits or prevents malignant cell phenotypes and/or which increases efficacy of an antimalignant therapeutic modality co-administered with the low dose NO mimetic. At this low dose, the known untoward effects of NO mimetics in animals without a malignant cell phenotype, cell, tumor and/or disease do not occur. As will be understood by those of skill in the art upon reading this disclosure, the nitric oxide mimetic increases, restores or maintains activity both in and around the cell (i.e. in the cellular microenvironment).

[0060] Methods for determining levels of nitric oxide of cells based upon nitrite, nitrate and S-nitrosothiol levels in cell culture, as well as plasma and serum, have been described. Serum or plasma nitrate levels in healthy normal volunteers have been reported to show a mean nitric oxide level of 33.4±8.9 μM with a range of 14 to 60 μM (Marzinzig et al. Nitric Oxide: Biology and Chemistry 1987 1(2): 177-189). These levels, however, are based on NO synthase end products, which accumulate and thus are likely to represent an overestimate of normal physiologic nitric oxide levels. Reported measured levels also vary depending upon the method selected for measurement. Further, levels of nitrite and nitrate in the plasma or serum are not solely representative of a patient's NO production. Based upon our experiments, we believe that normal physiologic levels of nitric oxide mimetic activity of cells may be lower, for example at least 5-fold, and preferably 10- to 10,000-fold lower, than those reported in the art, depending upon the cell.

[0061] Short-term nitric oxide mimetic therapy is generally administered at levels which increase nitric oxide mimetic activity of cells above normal physiologic levels. For purposes of the present invention, however, wherein longer-term therapy is generally desired, induction of tolerance against the NO mimetic and side effects become concerns. Thus, in certain aspects, the amount of nitric oxide mimetic administered is preferably very low so as to delay and/or reduce development of tolerance to the administered NO mimetic and/or unwanted side effects. For example, it is known that administration of nitric oxide or compounds which deliver nitric oxide to human beings at doses conventionally employed to treat cardiovascular conditions (i.e. GTN at 0.2 mg/h or greater) by vasodilation can provoke powerful vasodilator responses as well as development of drug tolerance against GTN upon repeated administration. Such administration is often accompanied by a number of undesirable side effects including headache, flushing and hypotension. In contrast, preferred doses of nitric oxide mimetic administered in the present invention to inhibit and prevent a malignant cell phenotype, cell, tumor and/or disease are lower, preferably at least 3 to 10,000-fold lower, more preferably at least 100- to at least 10,000-fold lower than those typically used in other therapeutic applications such as vasodilation and thus do not induce tolerance to the NO mimetic as quickly nor undesirable side effects. For example, using the nitric oxide mimetics sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), we have now demonstrated that amounts ranging between 10 ⁻¹² and 10 ⁻¹⁰ M in the cellular environment can be used to prevent and inhibit a malignant cell phenotype, cell, tumor and/or disease. Further, based on results from these experiments, we believe that doses of SNP as low as 10 ⁻¹⁴ M would be effective in preventing and inhibiting a malignant cell phenotype, cell, tumor and/or disease in less hypoxic or hyponitroxic environments. Table 1 provides additional examples of various lower preferred doses for nitric oxide mimetics useful in the present invention as well as the comparative higher doses used in vasodilation therapy.

[0062] As will be understood by those of skill in the art upon reading this disclosure, lower or higher amounts of nitric oxide mimetics than those exemplified herein can also be administered based upon the efficacy of the nitric oxide mimetic in achieving the ultimate goal of increasing, restoring or maintaining nitric oxide mimetic activity of cells so that a malignant phenotype is prevented or inhibited without substantial drug tolerance to the NO mimetic developing and without unwanted side effects. Determining amounts of nitric oxide mimetic to be incorporated into the low dose formulations of the present invention can be performed routinely by those skilled in the art based upon the teachings provided herein.

[0063] By the phrase “inhibiting and preventing” as used herein, it is meant to reduce, reverse or alleviate, ameliorate, normalize, control or manage a biological condition. Thus, inhibiting and preventing a malignant cell phenotype in accordance with the present invention refers to preventing development, reversing or ameliorating development and/or normalizing, controlling or managing development of a malignant cell phenotype, cell, tumor and/or disease. Additionally inhibiting and preventing a malignant tumor in accordance with the present invention refers to preventing development, reversing or ameliorating development and/or normalizing, controlling or managing development of a malignant tumor. Similarly, inhibiting and preventing a malignant disease in accordance with the present invention refers to preventing development, reversing the ameliorating development and/or normalizing, controlling or managing development of a malignant disease. Accordingly, administration of a low dose of a nitric oxide mimetic can be used both (1) prophylactically to inhibit and prevent a malignant cell phenotype, cell, tumor and/or disease from developing in animals at high risk for developing cancer or exposed to a factor known to decrease nitric oxide mimetic activity of cells, and (2) to treat cancer in animals by inhibiting metastases and development of resistance to antimalignant therapeutic modalities and increasing the efficacy of antimalignant therapeutic modalities. According to Stedman's Medical Dictionary, malignant is defined as 1) Resistant to treatment; occurring in severe form, and frequently fatal; tending to become worse and lead to an ingravescent course; and 2) in reference to a neoplasm, having the property of locally invasive and destructive growth and metastasis. In accordance with this definition, for purposes of the present invention, by “malignant cell phenotype” it is meant to encompass increases in metastasis, resistance to antimalignant therapeutic modalities, and angiogenesis. By “malignant cell phenotype, cell, tumor and/or disease” for purposes of the present invention, it is also meant to be inclusive of conditions in the spectrum leading to malignant behavior and abnormal invasiveness such as hyperplasia, hypertrophy and dysplasia, as well as those cells and tissue that facilitate the malignant process. Examples of conditions in this spectrum include, but are not limited to, benign prostatic hyperplasia and molar pregnancy. As evidenced by data presented herein, inhibition and prevention of a malignant cell phenotype in cells, tumors and/or diseases can be routinely determined by examining expression of genes including, but not limited to, uPAR, PSA, PAI-1, PROXY-1 and VEGF, by examining cell invasiveness in in vitro or in vivo assays and/or by examining resistance of the cells to antimalignant therapeutic modalities. It is believed that elevated phosphodiesterase expression and/or activity may be observed in cells with a malignant cell phenotype. Methods for measuring expression of these genes have been described for example in WO 99/57306, which is herein incorporated by reference. As will be understood by those of skill in the art upon reading this disclosure, however, other methods for determining gene expression via measurement of expressed protein or proteolytic fragments thereof can also be used. For purposes of the present invention, by the term “nitric oxide mimetic” it is meant nitric oxide, or a functional equivalent thereof; any compound which mimics the effects of nitric oxide, generates or releases nitric oxide through biotransformation, generates nitric oxide spontaneously, or spontaneously releases nitric oxide; any compound which in any other manner generates nitric oxide or a nitric oxide-like moiety or activates other stages of the NO pathway; or any compound which enables or facilitates NO utilization by the cell, when administered to an animal. Such compounds can also be referred to as “NO donors”, “NO prodrugs”, “NO producing agents”, NO delivering compounds”, NO generating agents”, NO releasing agents, and “NO providers”. Examples of such compounds include, but are not limited to: organonitrates such as nitroglycerin (GTN), isosorbide 5-mononitrate (ISMN), isosorbide dinitrate (ISDN), pentaerythritol tetranitrate (PETN), erthrityl tetranitrate (ETN); amino acid derivatives such as N-hydroxyl-L-arginine (NOHA), N ⁶ -(1-iminoethyl)lysine) (L-NIL), L-N ⁵ -(1-iminoethyl)ornithine (LN-NIO), N ^ω -methyl-L-arginine (L-NMMA), and S-nitrosoglutathione (SNOG);

Table 1

Typical Vasodilatory and Low Doses of Organonitrate

[0064] 1

[0065] NO prednisone and other compounds which generate or release NO under physiologic conditions such as S,S-dinitrosodithiol (SSDD), [N-[2-(nitroxyethyl)]-3-pyridinecarboxamide (nicorandil), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicilamine (SNAP), 3-morpholino-sydnonimine (SIN-1), molsidomine, DEA-NONOate(2-(N,N-diethylamino)-diazenolate-2-oxide), and spermine NONOate (N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl- 1,3-propanediamine). Organic nitrates GTN, ISMN, ISDN, ETN, and PETN, as well as nicorandil (commonly known as a potassium channel opener) are commercially available in pharmaceutical dosage forms. SIN-1, SNAP, S-thioglutathione, L-NMMA, L-NIL, L-NIO, spermine NONOate, and DEA-NONOate are commercially available from Biotium, Inc. Richmond, Calif. As used herein the term “nitric oxide mimetic” is also intended to mean any compound which acts as a nitric oxide pathway mimetic, that has nitric oxide-like activity, or that mimics the effect of nitric oxide. Such compounds may not necessarily release, generate or provide nitric oxide, but they have a similar effect to nitric oxide on a pathway that is affected by nitric oxide. For example, nitric oxide has both cyclic GMP-dependent and cyclic GMP-independent effects. Nitric oxide is known to activate the soluble form of guanylyl cyclase thereby increasing intracellular levels of the second messenger cyclic GMP and other interactions with other intracellular second messengers such as cyclic AMP. As such, compounds which directly activate either particulate or soluble guanylyl cyclase such as natriuretic peptides (ANP, BNP, and CNP), 3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole (YC-cGMP or YC-1) and 8-(4-chlorophenylthio) guanosine 3′,5′-cyclic monophosphate (8-PCPT-cGMP), are also examples of NO-mimetics. In some embodiments of the present invention, however, it is preferred that the NO-mimetic not encompass a compound which directly activates either particulate or soluble guanylyl cyclase. Nitric oxide mimetic activity encompasses those signal transduction processes or pathways which comprise at least one NO mimetic-binding effector molecule, such as for example, guanylyl cyclase and other heme containing proteins. Examples of agents which function as NO mimetics by enabling or facilitating NO utilization by the cell are compounds which inhibit phosphodiesterase activity and/or expression, such as phosphodiesterase inhibitors.

[0066] In a preferred embodiment of the present invention, more than one NO mimetic is administered. In this embodiment, it is preferred that the NO mimetics target or act upon different parts of the NO pathway of the cell. For example, an NO donor can be co-administered with a compound that inhibits cyclic nucleotide (e.g. cAMP or cGMP) degradation such as a phosphodiesterase inhibitor. Preferred phosphodiesterase (PDE) inhibitors useful as NO mimetics are those inhibiting PDE-1 through PDE-11.

[0067] By the term “hyponitroxia” in the present invention, it is meant conditions where levels of nitric oxide mimetic activity are lower than normal physiologic levels for that cell type.

[0068] Certain compounds suitable for use in the present invention are well known in the art and are described, e.g., in Goodman and Gilman, The Pharmacological Basis of Therapeutics (9th Ed.), McGraw-Hill, Inc. (1996); The Merck Index (12th Ed.), Merck & Co., Inc. (1996); The Physician 's Desk Reference (49th Ed.), Medical Economics (1995); and Drug Facts and Comparisons , Facts and Comparisons (1993).

[0069] NO Donors

[0070] In preferred embodiments, the compounds of the present invention are NO donors. The nitric oxide donor can be any of a variety of NO donors including, for example, organic NO donors, inorganic NO donors and prodrug forms of NO donors. Additional suitable NO donors include compounds that can be metabolized in vivo into a compound which delivers nitric oxide (e.g., a prodrug form of a NO donor; a NO-releasing drug such as a NO-releasing non-steroidal anti-inflammatory drug (NO-NSAIDs), examples of which include nitro-aspirin, NCX 4016, nitro-(flurbiprofen), HCT 1026, NCX 2216; or a binary NO generating system, such as acidified nitrates), or compounds that serve as physiological precursor of nitric oxide, such as L-arginine and salts of L-arginine. The NO donor may include at least one organic nitrate (including esters of nitric acid) and can be either a cyclic or acyclic compound. For example, suitable NO donors include nitroglycerin (NTG), isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN) which may include isosorbide-2-mononitrate (IS2N) and/or isosorbide-5-mononitrate (IS5N), erythrityl tetranitrate (ETN), pentaerythritol tetranitrate (PETN), ethylene glycol dinitrate, isopropyl nitrate, glyceryl-1-mononitrate; glyceryl-1,2-dinitrate, glyceryl-1,3-dinitrate, butane-1,2,4-triol trinitrate, and the like. Nitroglycerin and other organic nitrates including ISDN, ETN, and PETN, have been given regulatory approval for use in treatments in other fields of medicine on human subjects. Additional NO donors include sodium nitroprusside, N,O-diacetyl-N-hydroxy-4-chlorobenzenesulfonamide, N ^G -hydroxy-L-arginine (NOHA), hydroxyguanidine sulfate, molsidomine, 3-morpholinosydnonimine (SIN-1), (±)-S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), (±)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409), (±)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-y l]-3-pyridinecarboxamide (FR144420), and 4-hydroxymethyl-3-furoxancarboxamide. In addition, compounds that interfere with the breakdown of NO in vivo may be administered.

[0071] Additional NO Mimetics

[0072] In certain embodiments, the compounds and methods of the present invention are not limited to the foregoing traditional nitric oxide mimetics. As explained in detail below, these nitric oxide mimetics compounds include, for example, calcium channel blockers, α-adrenergic receptor antagonists and β-adrenergic receptor agonists, phosphodiesterase inhibitors, cAMP-dependent protein kinase activators, superoxide scavengers, potassium channel activators, benzodiazepines, adrenergic nerve inhibitors, antidiarrheal agents, HMG-CoA reductase inhibitors, adenosine receptor modulators, adenylyl cyclase activators, endothelin receptor antagonists, bisphosphonates, cGMP-dependent protein kinase activators, guanylyl cylase activators and SOC inhibitors. In certain aspects, the compounds are not limited to a low dose. Although a low dose can of course be used, the dosing of these compounds is not so limited. The compounds set forth below (e.g., PDE inhibitors) can be used at various doses such as high and low doses.

[0073] Calcium Channel Blockers

[0074] Ca ²⁺ channel blockers are compounds that inhibit the entry of Ca ²⁺ into the cell from the extracellular fluid. Suitable Ca ²⁺ channel blockers for use with the methods of the present invention include, but are not limited to, nifedipine, nimodipine, felopidine, nicardipine, isradipine, amlodipine, diltiazem, bepridil, verapamil etc. (see, e.g., WO 98/36733). L-type Ca ²⁺ channel blockers are also available.

[0075] α-adrenergic Receptor Antagonists and β-adrenergic Receptor Agonists

[0076] Additional preferred compounds for use in the context of the present invention include, e.g., α-adrenergic receptor antagonists and β-adrenergic receptor agonists. Suitable α-adrenergic receptor antagonists include, for example, α ₁ -adrenergic receptor antagonists, α ₂ -adrenergic receptor antagonists and other nonspecific α-adrenergic receptor antagonists. Preferred α ₁ -adrenergic receptor antagonists include, but are not limited to, prazosin, doxazosin, phenoxybenzamine, phentolamine, terazosin, tolazoline, etc., and are described in Goodman and Gilman, “ The Pharmaceutical Basis of Therapeutics,” 9th Edition, Hardman, et al. (ed.), McGraw-Hill (1996). Suitable α ₂ -adrenergic receptor antagonists include, but are not limited to, yohimbine and are also described in Goodman and Gilman, “ The Pharmaceutical Basis of Therapeutics,” 9th Edition, Hardman, et al. (ed.), McGraw-Hill (1996). Other suitable antagonists are α ₂ -adrenergic antagonists include, for example, post-synaptic α ₂ -adrenergic antagonists. These post-synaptic α ₂ -adrenergic antagonists include, but are not limited to, imiloxan, ARC 239 dihydrochloride and other pharmaceutically acceptable salts thereof. ARC 239 dihydrochloride is 2-[2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl]-4,4-dimethyl- 1,3-(2H,4H)-isoquinolindone dihydrochloride. Other suitable post-synaptic α ₂ -adrenergic antagonists include, but are not limited to, idazoxan, rauwolscine, efaroxan, mianserin, and mirtazapine. Of these, mianserin and mirtazapine are particularly preferred. Suitable β-adrenergic receptor agonists for use with the methods of the present invention include, but are not limited to, β ₁ -adrenergic receptor agonists, β ₂ -adrenergic receptor agonists, β ₃ -adrenergic receptor agonists and other nonspecific β-adrenergic receptor agonists. In a preferred embodiment, the β-adrenergic receptor agonist is a β ₂ -adrenergic receptor agonist or a β ₃ -adrenergic receptor agonists. Examples of β-adrenergic receptor agonists suitable for use with the methods of the present invention include, but are not limited to, albuterol, bitolterol, salbutamol, terbutaline, metaproterenol, procaterol, salmeterol, clenbuterol, isoproterenol, zinterol, BRL 37344, CL316243, CGP-12177A, GS 332, L-757793, L-760087, L-764646, and L-766892, etc. (see, e.g., Goodman and Gilman, supra).

[0077] Phosphodiesterase Inhibitors

[0078] In another preferred embodiment, the compound is a phosphodiesterase inhibitor. Cyclic nucleotide second messengers (cAMP and cGMP) play a central role in signal transductions and regulation of physiologic responses. Their intracellular levels are controlled by the complex superfamily of cyclic nucleotide phosphodiesterases (PDE) enzymes. Inhibitors of phosphodiesterases (PDE) are agents that can either activate or suppress PDEs via allosteric interaction with the enzymes or binding to the active site of the enzymes. The PDE family includes at least 19 different genes and at least 11 PDE isozyme families, with over 50 isozymes having been identified thus far. The PDEs are distinguished by (a) substrate specificity, i.e., cGMP-specific, cAMP-specific or nonspecific PDEs, (b) tissue, cellular or even sub-cellular distribution, and (c) regulation by distinct allosteric activators or inhibitors. PDE inhibitors include both nonspecific PDE inhibitors and specific PDE inhibitors (those that inhibit a single type of phosphodiesterase with little, if any, effect on any other type of phosphodiesterase). Still other useful PDE inhibitors are the dual selective PDE inhibitors (e.g., PDE III/IV inhibitors or PDE II/IV inhibitors). Below is a table setting forth various PDE inhibitors that are useful in the methods of the present invention. 2

[0079] In one embodiment, the PDE inhibitor is a PDE V inhibitor. Useful phosphodiesterase type V inhibitors include, e.g., cialis, vardenafil , tanadafil, zaprinast, MBCQ, MY-5445, dipyridamole, furoyl and benzofuroyl pyrroloquinolones, 2-(2-Methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8- (pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyridine- 3-carboxylic acid methyl ester hydrochloride (T-0156), T-1032 (methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridylmethoxy)-4-( 3,4,5-trimethoxy-phenyl)-3-isoquinoline carboxylate sulfate), and sildenafil. Cyclic GMP specific inhibitors include but not limited to A02131-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl thieno (3,2-c)pyrazole] for example. In another embodiment, the composition contains a phosphodiesterase type II (PDE II) inhibitor such as, e.g., EHNA. In yet another embodiment, the composition contains a phosphodiesterase type IV (PDE IV) inhibitor. Suitable phosphodiesterase type IV inhibitors include, but are not limited to, roflumilast, ariflo (SB207499), RP73401, CDP840, rolipram, mesopram, denbufylline, EMD 95832/3, cilomilast, RO-20-1724, and LAS31025. In still another embodiment, the phosphodiesterase inhibitor is a dual selective phosphodiesterase inhibitor such as, e.g., a PDE III/IV inhibitor (e.g., zardaverine) or phosphodiesterase inhibitors which can increase both cAMP and cGMP levels such as Satigrel (E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid).

[0080] In another embodiment, the PDE inhibitor is an inhibitor of the PDE III isozyme, for example, Olprinone.

[0081] In another embodiment, the PDE inhibitor is an inhibitor of the PDE IV isozyme family, or cAMP-specific and rolipram sensitive PDEs, which preferentially hydrolyze cAMP.

[0082] In yet another embodiment, the composition contains an agent that is a nonspecific phosphodiesterase inhibitor. Suitable nonspecific phosphodiesterase inhibitors include, but are not limited to, theobromine, dyphylline, IBMX, theophylline, aminophylline, pentoxifylline, papaverine, caffeine and other methylxanthine derivatives.

[0083] cAMP-Dependent Protein Kinase Activators

[0084] In other preferred embodiments, the compound used to treat the disorders described herein is a cAMP-dependent protein kinase activator. Examples of cAMP-dependet protein kinase activators include cAMP mimetics or dual cGMP/cAMP-dependent protein kinase activators. Suitable cAMP mimetics or analogs include those compounds that are structurally similar to cAMP and that have similar functions e.g., activities, as cAMP. Examples of suitable cAMP mimetics include, but are not limited to, 8-bromo-cAMP, dibutyryl-cAMP, Rp-cAMPS, and Sp-cAMPS, and useful dual activators include compounds such as, e.g., Sp-8-pCPT-cGMPS, Sp-8-bromo-cGMPS and 8-CPT-cAMP.

[0085] Superoxide Scavengers

[0086] In another embodiment, the compound used in the compositions and methods of the present invention is a superoxide anion (O ₂ ⁻ ) scavenger. Superoxide can react with NO and dramatically reduce its biological effects. Accordingly, agents that scavenge superoxide anions can enhance the effects of NO. Examples of superoxide scavengers include, but are not limited to, exogenous Mn or Cu/Zn superoxide dismutase (SOD) or small molecule SOD mimetics such as, e.g., Mn(III) tetra(4-benzoic acid) porphyrin chloride (MnTBAP) and M40403 (see, e.g., Salvemini, et al., Science, 286(5438):304-306 (1999)).

[0087] Potassium Channel Activators

[0088] In another aspect, the present invention provides pharmaceutical compositions comprising a potassium channel activator. In one embodiment, the potassium channel activator is an ATP-sensitive potassium channel activator. Synthetic compounds that activate ATP-sensitive K channels are smooth muscle relaxants. Such compounds include, but are not limited to, minoxidil, minoxidil sulfate, pinocidil, diazoxide, levcromokalim, cromokalim, etc. (see, e.g., White, et al, Eur. J. Pharmacol., 357:41-51 (1998)). Additional suitable ATP-sensitive K channel activators can be found in, e.g., Bristol, et al., “ Annual Reports in Medicinal Chemistry ,” Vol. 29, Chap. 8, pp. 73-82, Academic Press (1991). In another embodiment, the potassium channel activator is a Maxi-K channel activator. Examples of activators of the Maxi-K channels include, but are not limited to, estrogen-like compounds, such as estradiol (see, Valverde, et al., SCIENCE, 285:1929-1931).

[0089] Benzodiazepines

[0090] In another aspect, the present invention provides pharmaceutical compositions comprising a benzodiazepine. Suitable benzodiazepines include, but are not limited to, alprazolam, brotizolam, chlordiazepoxide, clobazam, clonazepam, chlorazepate, demoxepam, diazepam, estazolam, flumazenil, flurazepam, halazepam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam, prazepam, quazepam, temazepam, and triazolam (see, e.g., Goodman and Gilman, supra).

[0091] Adrenergic Nerve Inhibitors

[0092] In another aspect, the compounds of the present invention are compounds that inhibit adrenergic nerves. Adrenergic nerve inhibitors include compounds that destroy sympathetic nerve terminals, such as 6-hydroxydopamine and its analogs (see, e.g., Goodman and Gilman, supra). Adrenergic nerve inhibitors also include compounds that deplete norepinephrine storage, either by inhibiting norepinephrine biosynthesis or be depleting stores, and compounds that inhibit norepinephrine release. Compounds that inhibit norepinephrine biosynthesis include, but are not limited to, α-methyltyrosine. Compounds that deplete norepinephrine stores include, but are not limited to, reserpine, guanethidine and bretylium. Compounds that inhibit norepinephrine release include, but are not limited to, clonidine and other (α ₂ -adrenergic receptor antagonists. Examples of sympathetic nerve terminal destroyers include, but are not limited to, α ₂ -adrenergic receptor antagonists.

[0093] Antidiarrheal Agents

[0094] In another aspect, the compounds of the present invention are antidiarrheal agents. Examples of suitable antidiarrheal agents include, but are not limited to, diphenoxylate, loperamide, bismuth subsalicylate, octreotide, etc. (see, e.g., Goodman and Gilman, supra).

[0095] HMG-CoA Reductase Inhibitors

[0096] In another aspect, the compounds of the present invention are HMG-CoA reductase inhibitors. Examples of HMG-CoA reductase inhibitors include, but are not limited to, mevastatin, lovastatin, simvastatin, pravastatin, cerivastatin, dalvastatin, atorvastatin, fluvastatin, etc. (see, e.g., Goodman and Gilman, supra).

[0097] Smooth Muscle Relaxants

[0098] In other embodiments, the compounds of the present invention are smooth muscle relaxants such as, e.g., hydralazine, papaverine, tiropramide, cyclandelate, isoxsuprine and nylidrin.

[0099] Adenosine Receptor Modulators

[0100] In another aspect, the present invention provides compositions for the treatment for a malignant cell phenotype comprising adenosine receptor modulators, either alone or in combination with another agent. Methods for the use of these compositions are also provided. In one group of embodiments, adenosine receptor modulators are used alone. In another group of embodiments, the adenosine receptor modulators are combined with at least one other muscle-relaxing agent. In other embodiments, the compounds of the present invention are adenosine receptor modulators such as methylxanthines. Examples of adenosine receptor modulators include theophylline and dyphylline. For other examples see, Goodman & Gilman, supra). Preferred agents are selected from those described with reference to the compositions of single agents or combinations above.

[0101] Theophylline, a plant-derived methylxanthine, has been used for the treatment of bronchial asthma for decades. Theophylline relaxes smooth muscle, notably bronchial muscle, that has been contracted experimentally with a spasmogen, or clinically in asthma. Proposed mechanisms of methylxanthine-induced physiologic and pharmacological effects include: 1) inhibition of phosphodiesterases, thereby increasing intracellular cyclic AMP, 2) direct effects on intracellular calcium concentration, 3) indirect effects on intracellular calcium concentrations via cell membrane hyperpolarization, 4) uncoupling of intracellular calcium increases with muscle contractile elements, and 5) antagonism of adenosine receptors.

[0102] A related compound, i.e., dyphylline, is a preferred adenosine receptor modulator. This compound is not metabolized by the liver and is excreted unchanged by the kidneys, therefore its pharmacokinetics and plasma levels are independent of factors that affect liver enzymes such as smoking, age, congestive heart failure, or the use of other drugs that affect liver function.

[0103] Adenylyl Cyclase Activators

[0104] In another aspect, the present invention provides compositions comprising adenylyl cyclase activators, either alone or in combination with other compounds or agents described herein. The adenylyl cyclase activator forskolin is preferred. Other examples of adenylyl cyclase activators, include, but are not limited to, N6, O2′-dibutyryl-cAMP, 8-chloro-cAMP, and Rp-diastereomers of adenosine 3′,5′-cyclic monophosphorothioate, and related analogs, such as Rp-8-bromo-adenosine 3′,5′-cyclic monophosphorothioate, and derivatives of forskolin, including colforsin daropate hydrochloride.

[0105] Endothelin Receptor Antagonists

[0106] In another aspect, the present invention provides compositions comprising endothelin receptor antagonist, either alone or in combination with other compounds disclosed herein. Examples of endothelin receptor antagonists include, but are not limited to, BE 1827B, JKC-301, JKC-302, BQ-610, W-7338A, IRL-1038, LRL-1620, bosetan, ABT 627, Ro 48-5695, Ro 61-1790, tesosentan (Ro 61-0612, ZD1611, BMS-187308, BMS-182874, BMS-193884, sitaxsentan (TBC 11251), TBC 2576, TBC 3214, TBC-10950, ABT-627, atrasentan, A-192621, A-308165, A-216546, CI-1020, EMD 122946, J-104132 (L753037), LU 127043, LU 135252, LU 302872, TAK-044 (69), SB 209670, SB 234551, SB 247083, ATZ1993, PABSA, L-749,329, RPR111723, RPR11801A, PD 164800, PD 180988 (CI1034), IRL 3630, IRL 2500, and their derivatives, etc. (see, Doherty, Annual Reports in Medicinal Chemistry, Volume 35, pp. 73-82, Academic Press, 2000). Other ethenesulfonamide derivatives, which are endothelin receptor antagonists and which are useful in the methods of the present invention, are disclosed by Harada, et al., Chem. Pharm. Bull., 49(12):1593-1603 (2001).

[0107] Bisphosphonates

[0108] In another aspect, the present invention provides compositions comprising bisphosphonates, either alone or in combination with other agents. Suitable bisphosphonates suitable for use in the methods of the present invention include, but are not limited to, alendronate sodium (Fosamax), pamidronate disodium (Aredia), etidronate disodium Ididronel) and the like.

[0109] cGMP-Dependent Protein Kinase Activators

[0110] In another aspect, the present invention provides cGMP-dependent protein kinase activators, either alone or in combination with other agents disclosed herein. Suitable cGMP-dependent protein kinase activators include, but are not limited to, cGMP mimetics or dual cGMP/cAMP-dependent protein kinase activators. Suitable cGMP mimetics or analogs include those compounds that are structurally similar to cGMP and that have similar functions, e.g., activities, as cGMP. Examples of suitable cGMP mimetics include, but are not limited to, 8-bromo-cGMP, dibutyryl-cGMP, Rp-cGMPS, and Sp-cGMPS, and useful dual activators include compounds such as, e.g., Sp-8-pCPT-cGMPS, Sp-8-bromo-cGMPS and 8-CPT-cAMP.

[0111] Guanylyl Cylase Activators

[0112] For example, BAY 41-2272 is a novel non-NO-based direct stimulator of soluble guanylyl cyclase that activates purified enzyme in a synergistic fashion with NO.

[0113] SOC Inhibitors

[0114] In another aspect, the present invention provides store-operated calcium influx (SOC) inhibitors, which inhibit calcium uptake into non-excitable cells in response to stimulus-mediated depletion of intracellular calcium storage pools. The SOC inhibitors preferably inhibit one or more of the following: calcium-dependent activation of nuclear factor of activated T cells (NFAT), nuclear factor kB (NF-kB), the stress kinase c-Jun N-terminal kinase (JNK) and exocytosis, resulting in the release or elaboration of inflammatory mediators. Examples of SOC inhibitors include for example statins in the δ-lactone form such as lovastatin, mevastatin, fluvastatin, pravastatin, dalvastatin, cerivastatin, atrovastatin and simvastatin.

[0115] Other treatment options include NO mimetics in combination with an antioxidant to further enhance efficacy. Such antioxidants include for example, lycopene, resveratrol, green tea polyphenolics (e.g. ECGC), brassinin (from cruciferous vegetables like Chinese cabbage), sulforaphane (from broccoli) and its analog sulforamate, withanolides (from tomatillos), and n-acetyl cysteine.

[0116] For purposes of the present invention by the term “animal” it is meant to include all mammals, and in particular humans. Preferably, NO mimetics are administered to an animal at risk for or suffering from a malignant cell phenotype. Such animals are also referred to herein as subjects or patients in need of treatment.

[0117] Low oxygen levels have been correlated with an increased level of cellular invasion and invasiveness. Hypoxic stress causes a variety of cellular adaptations, often manifesting in the up-regulation of certain genes.

[0118] For example, it has been shown that uPAR mRNA and cell surface uPAR protein levels increase under hypoxic conditions. uPAR is a high affinity cell surface receptor for pro-urokinase-type plasminogen activator (pro-uPA). Upon binding of pro-uPA to uPAR, the inactive single-chain pro-uPA is cleaved into its active, two-chain form. The activated enzyme, still attached to the receptor, then acts to convert plasminogen into plasmin, which ultimately degrades several components of the extracellular matrix (ECM). Active uPA also serves to activate both latent metalloproteinases and growth factors. uPAR also serves as a receptor for the ECM molecule vitronectin and can also modulate integrin function. In combination, these functions increase cellular invasion and potential for invasiveness. A positive correlation between hypoxia-induced uPAR up-regulation and carcinoma cell invasiveness has been suggested (Graham et al. Int. J. Cancer 1999 80:617-623). In addition, we have now shown hyponitroxia induced by administration of the nitric oxide synthase antagonist L-NMMA (0.5 mM) in hypoxic (1% O ₂ ) and nonhypoxic (5% and 20% O ₂ ) conditions to increase uPAR mRNA levels in human MDA-MD-231 cells incubated for 24 hours at 37° C.

[0119] Although the role of uPAR in invasion and tumor progression has been studied extensively, the regulatory mechanisms governing its expression are poorly understood. The invasive potential of a cell is highly dependent upon its ability to penetrate the extracellular matrix (ECM) and basement membranes that impede its movement. This process involves the participation of a number of proteolytic enzyme systems, of which the uPA system figures prominently. We postulate that the cGMP-dependent regulation of invasion and metastasis seen the current study was at least partially mediated through alterations in uPAR expression.

[0120] We suggest a novel mechanism of oxygen sensing and uPAR regulation whereby phenotypes are modified in response to a decrease in cGMP-dependent signaling. This phenomenon is due to a reduction in endogenous NO synthesis. Molecular oxygen is obligatory for the conversion of L-arginine into NO and L-citrulline by the enzyme NO synthase (NOS). Indeed, exposure of cells to low levels of oxygen (1-3%) inhibits NO production by up to 90% and NOS inhibition has been shown to induce invasive phenotypes in a manner similar to hypoxia. Due to the reduced NO levels, there is a decrease in GC activity and a consequential reduction in cellular cGMP. Indeed, Taylor et al. (1998) showed that culturing intestinal epithelial cells in hypoxia (1% O ₂ ; 24 Hrs) resulted in a significant decrease in basal and stimulated cGMP levels. Our studies have also suggested that a potential oxygen sensing mechanism of epithelial cells involves the participation of heme containing proteins such as GC. We have similarly shown that low oxygen levels decrease cGMP generation, and that they also result in phenotypic alterations. In both studies, these alterations were overcome by the addition of 8-Br-cGMP. We have further shown that inhibition of soluble guanylyl cyclase (sGC) with 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ) can antagonize the ability of NO-mimetics to prevent the hypoxic up-regulation of uPAR. Given these findings we have derived a working model for the oxygen-mediated regulation of uPAR ( FIG. 4 ). In this model, low oxygen levels result in decreased NO synthesis and a consequential decrease in sGC activation. As such, cellular cGMP levels are lowered, resulting in decreased PKG activity.

[0121] One possible mechanism involves a perturbation of the MAP kinase signaling pathway. This pathway is activated by hypoxia and studies have shown that NO can prevent the phosphorylation of ERK through a PKG-mediated interference of RAS/Raf. This concept has been strengthened by Yoshihide et al. (2000) who showed that NO donors and cGMP mimetics could reduce elastase levels by suppressing ERK phosphorylation. This led to a subsequent reduction in the activation and DNA binding capacity of AML1B (the transcription factor for elastase). It is possible that cGMP-dependent NO signaling similarly inhibits hypoxia-induced ERK phosphorylation, thereby decreasing the activation of the transcription factors responsible for the up-regulation of uPAR.

[0122] The promotor region of uPAR contains binding sites for transcription factors such as activator protein-1 (AP-1), Sp-1/3 and nuclear factor κB (NFκB). Hypoxia Inducible Factor 1 (HIF-1) levels may also contribute to the transcriptional activation of the uPAR gene, as previous examination of the sequence upstream of the uPAR initiation codon revealed the presence of at least 3 potential HIF-1-binding sites. It has also been shown that HIF-1 accumulation and transcriptional activity can be reduced by relatively high concentrations (2.5-500 μM) of NO mimetics such as SNP, S-nitroso-L-glutathione and 3-morpholinosydnonimine. Preliminary studies in our laboratory have confirmed that high concentrations (0.1-1 mM) of GTN and SNP inhibit HIF-1 accumulation and transactivating activity. However, concentrations of the NO mimetics that completely inhibited the hypoxic stimulation of metastasis, invasiveness and uPAR expression had no effect on HIF-1 accumulation or transactivating activity under hypoxia. Similarly, the administration of 8-Br-cGMP did not affect HIF-1 activity. Therefore it is unlikely that the cGMP-dependent regulation of invasive phenotypes is mediated via alterations in HIF-1.

[0123] Like uPAR, PAI-1 has also been shown to be stimulated under hypoxic conditions. See WO99/57306. Further, this stimulation was accompanied by a decrease in cellular adherence. PAI-1 is 52-kDa ECM glycoprotein which is produced by a variety of normal and malignant cells. This glycoprotein is a regulator of plasminogen activator activity. It functions to inhibit both free and bound uPA through the formation of irreversible covalent complexes. PAI-1 has also been shown to compete with the uPAR for binding to the same domain of vitronectin. As such, PAI-1 is capable of releasing cells bound to vitronectin-coated plates. Studies have shown that PAI-1 is required for the optimal in vitro invasiveness of lung carcinoma cells.

[0124] Hypoxia has also been shown to increase the resistance of cells to cytotoxic agents. The gene for PROXY-1 was identified using an RT-PCR based differential display following the culture of a variety of cell types under low levels of oxygen. See WO99/57306. It is believed that the 43-kDa PROXY-1 protein plays a role in protecting cells from insults including hypoxia, DNA damaging agents, cytotoxic agents and glucose deprivation, as enhanced PROXY-1 expression is observed in response to each of these harmful stimuli. Together with the fact that this gene is expressed by a variety of unrelated cell types, this type of gene expression is indicative of PROXY-1 being a universal ‘switch’ involved in the initial events that lead to cellular adaptations to hypoxia.

[0125] However, it is possible that the role of NO in tumor cell initiation promotion, progression, survival and apoptosis depends on the cell types, the concentrations of NO in the given cellular microenvironment, time of cellular exposure to NO, and possibly other factors. A similar paradigm has been drawn from the role of NO in acute and chronic inflammatory processes and in myocardial and neuro-protective preconditioning, for example. As a plieotropic transcriptional factor, NFκB (nuclear factor kappa B)-induced antiapoptotic signals have been suggested to be responsible for the development of chemoresistance in various forms of cancer, in tumor progression, and in the development of radiation resistance. NF-κB as a molecular target for developing anti-cancer therapy has been reviewed extensively by several investigators. Various known NFκB inhibitors such as NSAIDs, glucocorticoids, COX 2 inhibitors, and more recently proteasome inhibitors (blocks NF-κB activation), have been shown to be effective or potentially effective cancer treatment options. Known NFκB inducible genes that may be involved in tumor progression and chemoresistance include VEGF (vascular endothelium growth factor), EGFR (epidermal growth factor receptor), COX2 (cyclooxygenase type 2), MMPs (matrix metalloproteases 2 and 9 for example), uPAR (urokinase plasminogen activator receptor), etc. In a recent review article on inflammation and cancer, anti-inflammatory therapy is considered an efficacious approach towards early neoplastic progression and malignant conversion

[0126] It is possible that NO may function as the feedback inhibitor of NFκB upregulation/activation. Alternatively, NO may affect p53 tumor suppressor gene expression, or BcL expression. It is also possible that NO may enhance chemosensitivity of tumor cells via a completely unknown mechanism. While the traditional approach to treat cancer with combination chemotherapeutic agents and/or radiation therapy is primarily based on the known mechanism of action, current approach focuses on the balancing the effectiveness of the treatment.and toxicity profile of selected drugs. Since many of the NO mimetics are proven to be safe for both acute and chronic usage, it presents as a unique opportunity to minimize the safety risk when NO mimetics is added to the known standard of treatments of various forms of cancer.

[0127] In some forms of cancer, for example, prostate and breast cancers, hormonal therapy is typically used for early phase of the diseases. Hormonal therapy includes anti-androgens (e.g. flutamide) for prostate cancer and anti-estrogen (e.g. tamoxifen) for estrogen receptor positive or ER status unknown early stage breast cancers. In general, the tumor responded well to the hormonal therapy before a new phenotype is developed. The newly developed phenotypes are typically no longer responding to the original hormonal therapy; prognosis for patients at this stage of cancer, i.e. hormone refractory/insensitive phase is typically very poor. Even while using hormonal therapy during the hormone responding phase of cancers, patients suffer from hot flashes, loss of libido, sexual dysfunction, osteopenia, osteoporosis, poor self-esteem and quality of life. The mechanism leading to the loss of hormone response of these cancer phenotypes are largely unknown at this point. It could be related to androgen/estrogen receptor expression levels, cellular locations, functional activities of these steroid receptors. Alternatively, it could also due to the altered non-genomic actions of these steroid targets at various cellular compartments and sub-compartment, e.g. mitochondria.

[0128] Endogenous androgens including testosterone and 5-α-dihydrotestosterone (DHT; one of the two key metabolites of testosterone) are essential for the development and maintenance of reproductive tissues (e.g. testis, epididymis, seminal vesicles, penis, etc.) in male species. Exogenously administered androgens, e.g. testosterone undecanoate, testosterone propionate, etc. typically transformed to testosterone or DHT before exerting its biological activity. The majority of the biological activities of testosterone and DHT are elicited by the cytosolic androgen receptor (AR). The AR is a member of the steroid hormone-thyroid hormone-retinoic acid nuclear receptor super-family. Upon androgen binding, the AR undergoes conformational changes resulting in the release of inhibitory proteins and is subsequently hyper-phosphorylated, translated to the cell nucleus. Once in the nucleus, AR is dimerized and binds to hormone response elements in the regulatory regions of androgen target genes, and subsequent gene transcriptional events. DHT binds to AR with higher affinity and induces a higher level of androgen-regulated gene expression than testosterone. Since the second key metabolite is E ₂ , the biological activity of testosterone is also mediated at least in part, by the activation of estrogen receptor.

[0129] Evidence has been recently obtained for ligand-independent activation of AR transcriptional activity by peptide growth factors, such as IGF-I (insulin-like growth factor), and to a lesser extent, keratinocyte growth factor and epidermal growth factor. Like other steroid receptor ligands, testosterone can also elicit biological activities via non-genomic mechanisms that often involve ion fluxes in androgen-dependent cell types. Specifically, calcium fluxes following addition of picomolar concentrations of testosterone have been reported in rat heart myocytes, male rat osteoblasts, human prostate cancer cells, and Sertoli cells. Direct evidence from patch-clamp studies showed that testosterone can open K ⁺ -channels in single coronary myocytes. Although testosterone mediated ion fluxes have been reported in various cell types, the biological consequences of these activities are still poorly defined.

[0130] While infrequent in early and localized prostate tumors, mutations of the AR have been identified in a number of advanced prostatic cancers. Substantial evidence suggests that AR activity is enhanced in advanced forms of prostate cancer, and in some cases, as a result of gene amplification events. Frequently, the AR also appears to have altered ligand specificity; for example, advanced prostate cancers often become androgen-independent, and no longer respond to androgen ablation therapy. The loss of androgen-sensitivity is considered an indicator of poor prognosis for advanced prostate cancers.

[0131] NO mimetics may be able to maintain the tumor cell under homeostatic stage, thus prevent the alteration of steroid receptor expressions, levels of expression, location, or alternatively, the ion-channels related to androgen and anti-androgen action etc. Thus, the administration of NO mimetics could keep patients under hormonal therapy for various forms of cancer under hormone responsive phase, preventing or delaying metastasis to secondary sites and transformation to more advanced, hormone-refractory/insensitive/insensitive cancers. NO mimetics can be administered in conjunction with hormonal therapy during the treatment phase and/or can be used in the remission phase.

[0132] In certain cases, NO mimetics can be used with chemo-and/or radio-therapeutic treatment to ensure eradication