WO2003074082A1 - Formulations et procedes d'utilisation de substances mimetiques de l'oxyde nitrique dans le traitement du cancer - Google Patents

Formulations et procedes d'utilisation de substances mimetiques de l'oxyde nitrique dans le traitement du cancer Download PDF

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WO2003074082A1
WO2003074082A1 PCT/CA2003/000313 CA0300313W WO03074082A1 WO 2003074082 A1 WO2003074082 A1 WO 2003074082A1 CA 0300313 W CA0300313 W CA 0300313W WO 03074082 A1 WO03074082 A1 WO 03074082A1
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nitric oxide
cells
cancer
mimetic
tumor
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PCT/CA2003/000313
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English (en)
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Charles H. Graham
Lynne-Marie Postovit
Michael A. Adams
Jeremy P. W. Heaton
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Cellegy Pharmaceuticals, Inc.
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Priority to CA002478145A priority Critical patent/CA2478145A1/fr
Priority to EP03706181A priority patent/EP1492567A1/fr
Priority to JP2003572598A priority patent/JP2005527510A/ja
Priority to AU2003208228A priority patent/AU2003208228A1/en
Publication of WO2003074082A1 publication Critical patent/WO2003074082A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to methods and formulations for inhibiting, treating and preventing a malignant cell phenotype, cell, tumor and/or disease.
  • hypoxia and hyponitroxia have impact upon cellular phenotype is not necessarily mediated solely by the lack of oxygen but rather also from a deficiency in nitric oxide mediated signaling.
  • administration of nitric oxide mimetics, such as low doses is sufficient to increase, restore or maintain nitric oxide-mediated signaling in cells so that malignant cell phenotypes, cells, tumors and/or diseases are inhibited or prevented.
  • These methods and formulations are particularly useful in treating and preventing cancer in animals.
  • hypoxia affects endothelial cellular physiology in vivo and in vitro in various ways including modulating the transcriptionally-regulated expression of vasoactive substances and matrix proteins involved in modulating vascular tone or remodeling the vasculature and surrounding tissue (Faller, DN. Clin. Exp. Pharmacol and Physiol. 1999 26:74-84).
  • Hypoxia in solid tumors has been shown to protect cancer cells from being killed by X-irradiation and leads to resistance to certain cancer drugs. Hypoxia also appears to accelerate malignant progression and increase metastasis (Brown, J.M. Cancer Res. 1999 59:5863-
  • Nitric oxide has been implicated in various biological processes. For example, nitric oxide is a biological messenger molecule responsible for endothelium derived vascular relaxation and neurotransmission. Nitric oxide, at what these researchers refer to as high levels, is also known as a mediator for anti-tumor and anti-bacterial actions of macrophages. Nitric oxide has also been demonstrated to play a modulatory role on cytokine-induced expression of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases (Eberhardt et al. Kidney International 2000 57:59-69).
  • Soluble guanylyl cyclase sGC is the best defined downstream target of NO.
  • NO is a pleiotropic molecule
  • sGC guanosine 5 '-triphosphate
  • cGMP cyclic guanosine monophosphate
  • Nitric oxide binds to the heme moiety of sGC, inducing a number of conformational changes that result in its activation.
  • cGMP is a potent second messenger that allows NO to transmit its signal to downstream effectors. Elevated levels of cGMP have been negatively correlated with vascular smooth muscle growth and have been shown to prevent platelet aggregation as well as the adherence of neutrophils to endothelial cells. Further, cGMP levels play a role in gene regulation. A cGMP analogue (8-Br-cGMP) has been shown to prevent the hypoxic induction of NEGF. The endogenous inhibition of endothelin-1 (ET-1) by NO is also, . mediated through a guanylate cyclase/cGMP- dependent mechanism. [07] There are several cGMP target proteins that could mediate NO-regulated gene expression.
  • PKG cGMP-dependent protein kinase
  • PDEs cGMP-activated phosphodiesterases
  • cGMP-gated ion channels cGMP-gated ion channels
  • cAMP-dependent protein kinases cGMP-dependent protein kinase
  • PKG is responsible for the majority of cGMP's intracellular actions.
  • PKG is a serine/threonine protein kinase that is selectively activated by cGMP binding.
  • PKG phosphorylates many intracellular targets, often resulting in alterations in gene expression. For example, PKG has been shown to modulate the transcription of various genes through its activation of factors such as AP-1 and the serum response element.
  • nitric oxide plays a role in promoting solid tumor growth and progression.
  • nitric oxide generation by inducible nitric oxide synthase (iNOS) has been implicated in the development of prostate cancer (Klotz et al. Cancer; National Library of Medicine, MDX Health Digest 1998
  • nitric oxide has been suggested to play an important role in the metabolism and behavior of lung cancers, and in particular adenocarcinomas (Fujimoto et al. Jpn. J. Cancer Res 1997 88:1190-1198).
  • tumor cells producing or exposed to what these researchers refer to as low levels of nitric oxide, or tumor cells capable of resisting nitric oxide-mediated injury undergo a clonal selection because of their survival advantage (Lala, P.K. and Orucevic, A. Cancer and Metastasis Review 1998 17:91-106).
  • tumor-derived nitric oxide promotes tumor angiogenesis as well as invasiveness of certain tumors in animals, including humans (Lala, P.K. Cancer and Metastasis Reviews 1998 17:1-6).
  • nitric oxide has been reported to reverse production of vasoconstrictors induced by hypoxia (Faller, D.G. Clinical and Experimental Pharmacology and Physiology 1999 26:74-84).
  • the nitric oxide donor sodium nitroprusside (SNP; 150 ⁇ M) has also been demonstrated to decrease hypoxia-induced expression of vascular endothelial growth factor, an endothelial cell mitogen required for normal vascular development and pathological angiogenie diseases such as cancer and iris and retinal neovascularization (Ghiso et al. Investigative Ophthalmology & Visual Science 1999 40(6):1033-1039).
  • 150 ⁇ M SNP was demonstrated to completely suppress hypoxia-induced NEGF mR ⁇ A levels for at least 24 hours in immortalized human retinal epithelial cells.
  • nitric oxide when induced in certain cells, can cause cytostasis and apoptosis.
  • Xie et al. have demonstrated exposure to high levels of nitric oxide (producing approximately 75 ⁇ M nitrite; see Figure 5 A of Xie et al.) to be an exploitable phenomenon to promote death (see Figure 6 A and 6B of Xie et al.) in murine K-1735 melanoma cells (J. Exp. Med. 1995 181:1333-1343).
  • WO 93/20806 discloses a method of inducing cell cytostasis or cytotoxicity by exposing cells to a compound such as spermine-bis(nitric oxide) adduct monohydrate at 500 ⁇ M which is capable of releasing nitric oxide in an aqueous solution.
  • a compound such as spermine-bis(nitric oxide) adduct monohydrate at 500 ⁇ M which is capable of releasing nitric oxide in an aqueous solution.
  • the compounds are taught to be useful in the treatment of tumor cells as well as in antiparasitic, antifungal and antibacterial treatments.
  • a mega-dosing regimen wherein a large dose of the nitric oxide releasing compound is administered, time is allowed for the active compound to act, and then a suitable reagent such as a nitric oxide scavenger is administered to the individual to render the active compound inactive and to stop nonspecific damage.
  • U.S. Patent 5,840,759, U.S. Patent 5,837,736, and U.S. Patent 5,814,667 disclose methods for using mg/kg quantities of nitric oxide releasing compounds to sensitize hypoxic cells in a tumor to radiation. These patents also disclose methods of using the same nitric oxide-releasing compounds at mg/kg levels to protect non-cancerous cells or tissue from radiation, to sensitize cancerous cells to chemotherapeutic agents, and to protect non-cancerous cells or tissue from chemotherapeutic agents. Compounds used in these methods spontaneously release nitric oxide under physiologic conditions without requiring oxygen. These patents teach administration of the nitric oxide-releasing compound from about 15 to about 60 minutes prior to therapy.
  • Typical doses of the nitric oxide releasing compound administered are suggested to be from about 0.1 to about 100 mg of one or more nitric oxide releasing compounds per kg of body weight.
  • Concentrations of the nitric oxide releasing compounds DEA/NO and PAPA/NO demonstrated to increase the sensitivity of MCF7 breast cancer cells and N79 fibroblasts to melphalan, thiotepa, mitomycin C, SR4233 and cisplatin in vitro were in the millimolar range while 70 mg/kg of DEA/ ⁇ O was demonstrated to increase the survival of mice administered the chemotherapeutic agent melphalan in the in vivo KHT tumor model.
  • U.S. Patent 5,700,830 and WO 96/15781 disclose methods for inhibiting adherence between cancerous cells and non-cancerous cells in an animal by administering to the animal a nitric oxide-releasing compound containing a nitric oxide-releasing ⁇ 2 O 2 functional group.
  • studies indicate that cancer cell adhesion to and spreading along the vessel wall leading to extravasation is not an obligatory event in metastasis
  • WO 98/58633 discloses a microdose nitric oxide therapy for alleviating vascular conditions associated with a reduction in nitric oxide production or an attenuation of nitric oxide effect.
  • the present invention provides methods and formulations for administering nitric oxide mimetics to cells to inhibit, to treat and to prevent a malignant cell phenotype, cell, tumor and/or disease.
  • the nitric oxide mimetic is administered in a low dose.
  • the nitric oxide mimetics of the present invention increase the efficacy of palliative treatment, by relieving or reducing the intensity of uncomfortable symptoms associated with cancer, such as pain, nausea, and shortness of breath.
  • the present invention provides a method useful for controlling, treating, and/or preventing cancer, malignancies, neoplasm, hyperplasia, hypertrophy, dysplasia and/or tumor angiogenesis, comprising administering a nitric oxide mimetic to an animal to control, treat and/or prevent cancer, malignancies, neoplasm, hyperplasia, hypertrophy, metastasis, dysplasia and/or tumor angiogenesis.
  • the nitric oxide mimetic is administered at a low dose.
  • the nitric oxide mimetic is administered at a level which delays and/or reduces development of tolerance to the nitric oxide mimetic and/or unwanted side effects, including headache, flushing, syncope, dizziness and hypotension.
  • the nitric oxide mimetic is administered alone or in combination with an antimalignant therapeutic agent.
  • the nitric oxide mimetic is administered in combination with agents intended for palliative purposes including pain relief, improving physical strength and quality of life.
  • the nitric oxide mimetic (1) inhibits the metastatic potential of a malignant cell phenotype, cell, tumor and/or disease preferably by decreasing the invasiveness, progression, growth and/or metastases of cells, tumors and/or diseases exhibiting a malignant phenotype; inhibiting the survival and/or growth of cells, tumors and/or diseases exhibiting a malignant phenotype; decreasing the progression and/or metastases of cells, tumor volume and/or diseases exhibiting a malignant phenotype; increasing the regression of cells, tumors and/or diseases exhibiting a malignant phenotype; and/or facilitating the killing of cells, tumors and/or diseases exhibiting a malignant phenotype; (2) maintains a malignant cell, tumor and/or disease in a dormant state at its primary and/or secondary site; (3) enhances the efficacy of, increases sensitivity to, and/or prevents or decreases the resistance of a malignant cell, tumor and/or disease to an
  • the cells exhibiting the malignancies are selected from malignant cells, invasive cells, cells and tissue(s) that facilitate the malignant process, and combinations thereof; optionally wherein the malignant cell phenotype is controlled, treated or prevented by improving response to an antimalignant therapeutic modality.
  • the nitric oxide mimetic (1) inhibits or retards the metastasis of existing tumors in a patient preferably by decreasing the invasiveness, progression, growth and/or metastases of existing tumor(s); inhibiting the survival and/or growth of existing tumor(s); decreasing the progression and/or metastases of existing tumor(s); increasing the regression of existing tumor(s); and/or facilitating the killing of existing tumor(s); (2) maintains a malignant tumor in a dormant state at its primary and/or secondary site; (3) enhances the efficacy of, increases sensitivity to, and/or prevents or decreases the resistance of tumor(s) to an antimalignant therapeutic modality; or (4) inhibits or prevents tumor angiogenesis in animals at high risk of developing cancer and/or exposed to factors known to decrease nitric oxide activity in an animal, optionally wherein said factors include decreased arginine levels, exposure to nitric oxide synthase antagonists, exposure to nitric oxide scavengers, changes
  • the nitric oxide mimetic (1) inhibits or retards the metastasis of a cancerous disease in a patient preferably by decreasing the invasiveness, progression, growth and/or metastasis of the disease; inhibiting the survival and/or growth of the disease; decreasing the progression and/or metastasis of the disease; increasing the regression of the disease; and/or facilitating the curing and/or treatment of the disease; (2) produces or maintains remission of the disease; (3) enhances the efficacy of, increases sensitivity to, and/or prevents or decreases the resistance to an antimalignant therapeutic modality used to treat the disease; or (4) inhibits or prevents development of the disease in animals at high risk of developing cancer and/or exposed to factors known to decrease nitric oxide activity in an animal, optionally wherein the factors include decreased arginine levels, exposure to nitric oxide synthase antagonists, exposure to nitric oxide scavengers, changes in nitric oxide synthase expression, change in
  • the cells exhibiting the malignancies are selected from malignant cells, invasive cells, cells and tissue(s) that facilitate the malignant process, and combinations thereof; optionally wherein the malignant cell phenotype is controlled, treated or prevented by improving response to an antimalignant therapeutic modality.
  • cancer is diagnosed or monitored by measuring a tumor selective marker present in the animal.
  • the nitric oxide mimetic decreases, maintains, or decelerates increases of the level of the tumor marker.
  • the cancer comprises gastric cancer, gastrointestinal cancer, testicular cancer, prostate cancer, prostatie adenocarcinoma, breast cancer, metastatic melanoma, lung cancer, the cancers set forth in Table 2 or combinations thereof.
  • the cancer or other malignancies, neoplasm, hyperplasia, hypertrophy, dysplasia and/or tumor angiogenesis in an animal comprises benign prostatie hyperplasia or molar pregnancy.
  • the nitric oxide mimetic comprises nitric oxide, a nitric oxide donor, a nitric oxide prodrug, a compound that generates or releases nitric oxide through biotransformation, a compound that generates nitric oxide spontaneously or spontaneously releases nitric oxide, or a compound which generates nitric oxide, or combinations thereof.
  • the nitric oxide mimetic is (1) a nitric oxide donor selected from nitroglycerin (GTN), isosorbide 5-mononitrate (ISMN), isosorbide dinitrate (ISDN), pentaerythritol tetranitrate (PETN), erthrityl tetranitrate (ETN), N-hydroxyl-L- arginine (NOHA), N 6 -(l-iminoethyl)lysine) (L-NIL), L-N 5 -(l-iminoethyl) ornithine (LN- NIO), N w -methyl-L-arginine (L-NMMA), S-nitrosogluthathione (SNOG), S, S- dinitrosodithiol (SSDD), [N-[2 (nitroxyethyl)]-3-pyridinecarboxamide (nicorandil), sodium nitroprusside (SNP), S
  • GTN nitro
  • spermine NONOate N-[4-[l-(3-aminopropyl)-2-hydroxy-2- nitrosohydrazino]butyl-l, 3-propanediamine; (2) a compound that activates stages of NO pathway, a compound which enables or facilitates NO utilization by a cell, a compound which directly activates guanylyl cyclase, or a phosphodiesterase inhibitor, or combinations thereof; (3) a non-specific phosphodiesterase inhibitor, a dual-selective phosphodiesterase inhibitor, a type I, II, III, IN, N, NI, Nil, NIII, IX, X, or XI phosphodiesterase inhibitor, or combinations thereof; or (4) a protein kinase G activator.
  • the antimalignant therapeutic agent includes radiation therapy (radiotherapy), thermal therapy, immunotherapy, hormonal therapy, or single agent chemotherapy, combination chemotherapy, chemo-irradiation, adjuvant therapy, neoadjuvant therapy, palliative therapy or combinations thereof.
  • the antimalignant therapeutic modality comprises radiation therapy
  • the nitric oxide mimetic is a nitric oxide, a nitric oxide donor, a compound that generates or releases nitric oxide through biotransformation, or a compound that generates nitric oxide spontaneously or spontaneously releases nitric oxide only in the presence of oxygen, or combinations thereof, wherein the nitric oxide mimetic is administered during the radiation therapy.
  • the chemotherapy comprises administration of chemotherapeutic agent that is an anti-angiogenic agent, an antimetabolite, an antibiotic, an endothelin activating agent, an enzyme inhibitor, a hormonal agent, ocreotide acetate, a microtubule-disruptor agent, a microtubule-stabilizing agent, a vinca alkaloid, an epipodophyllotoxin, a topoisomerase inhibitor; a prenyl-protein transferase inhibitor, hydroxyurea, procarbazine, mitotane, hexamethylmelamine, a platimum coordination complex, a biological response modifier, a growth factor, an immune modulator, or a monoclonal antibody, or a combination thereof.
  • chemotherapeutic agent that is an anti-angiogenic agent, an antimetabolite, an antibiotic, an endothelin activating agent, an enzyme inhibitor, a hormonal agent, ocreotide acetate, a microtubule-disrupt
  • the palliative therapy comprises glucocorticoid steroids, narcotic pain relief agents, anti-depressants, sex hormones, or a combination thereof.
  • the dose of nitric oxide mimetic is at least 3- to 10,000- fold lower, preferably 100-10,000 fold lower than a dose of nitric oxide mimetic that produces vasodilation.
  • the nitric oxide mimetic is an organic nitrate and the mimetic is administered at a dose of at least 3- to 10,000-fold lower, preferably 100-
  • the nitric oxide mimetic is a known vasodilatory compound and the mimetic is administered at a dose of at least 3- to 10,000-fold lower, preferably 100-10,000 fold lower than the dose of nitric oxide mimetic known to produce vasodilation
  • the nitric oxide mimetic is selected from the group consisting of a calcium channel blocker, an ⁇ -adrenergic receptor antagonist, a ⁇ - adrenergic receptor agonist, a phosphodiesterase inhibitor, a cAMP-dependent protein kinase activator, a superoxide scavenger, a potassium channel activator, a benzodiazepine, an adrenergic nerve inhibitor, an antidiarrheal agent, a HMG-CoA reductase inhibitor, an adenosine receptor modulator, a adenylyl cyclase activator, an endothelin receptor antagonist, a bisphosphonate, a cGMP-dependent protein kinase activator, a guanylyl cyclase activator and a SOC inhibitor.
  • a calcium channel blocker an ⁇ -adrenergic receptor antagonist, a ⁇ - adrenergic receptor agonist,
  • methods and formulations are particularly useful in controlling cancer by reducing its growth and improving response to therapy.
  • methods and formulations of the present invention can inhibit metastasis, invasiveness and progression of cells, tumors and/or diseases exhibiting a malignant phenotype.
  • the methods and formulations can induce or maintain dormancy or remission of cells, tumors and/or diseases exhibiting a malignant phenotype at primary as well as secondary sites.
  • these methods and formulations can prevent or decrease development of resistance of cells, tumors and/or disease exhibiting a malignant phenotype to antimalignant therapeutic modalities, as well as increase the efficacy of antimalignant therapeutic modalities.
  • the methods and formulations of the present invention are also very useful in preventing a malignant cell phenotype, cell, tumor and/or disease that can develop upon exposure of cells to conditions and/or therapeutic agents which lead to a deficiency in nitric oxide mimetic activity in the cells.
  • the methods and formulations of the present invention are also useful in inhibiting development of a more aggressive malignant cell phenotype in cancer cells, tumors, and/or diseases, which can occur upon exposure to factors which induce such development.
  • these methods and formulations are useful in diagnosing and monitoring a malignant cell phenotype, cell, tumor and/or disease in an animal via detection of levels of one or more markers indicative of a malignant phenotype following administration of a low dose of a nitric oxide mimetic. 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 phenotype in the animal. Accordingly, the methods and formulations of the present invention provide new therapeutic and diagnostic approaches for the treatment and prevention of cancer in animals.
  • the compounds, and methods described herein prolong cancer remission, prevent the recurrence of cancer, decrease cancer markers, reduce tumor volume, reduce pain, discomfort, and disability (morbidity), increase the quality of life associated with antimalignant therapeutic modalities, decrease cachexia, and reduce the need for anti-emetic agents and narcotic pain killers.
  • Figure 1 is a histogram showing the effect of GTN and SNP on the in vitro invasion by MDA-MB-231 invasive breast cancer cells in hypoxic (1% O 2 ) conditions as compared to normal (20% O 2 ) conditions.
  • Cells were coated onto Matrigel-coated membranes and incubated under hypoxic or normal conditions, alone or in the presence of nitric oxide mimetics.
  • the invasion index (% of control) which is taken to be a measure of the invasive potential of the cells for each treatment, was determined by staining the cells that invaded through the membrane and counting them.
  • the first bar depicts the invasion index of cells cultured under normal conditions (20% O 2 ).
  • the second bar depicts the invasion index of cells cultured under hypoxic conditions (1% O 2 ).
  • the third bar depicts the invasion index of cells cultured under hypoxic conditions (1%
  • Figure 2 is a histogram showing the lung colonization ability of B 16F 10 mouse melanoma cells incubated for 12 hours in 1% or 20% O 2 in the presence or absence of 2 x 10 "11 M (20 pM) GTN and injected i.v. (tail vein) into C57B16 female mice. Fourteen days later, mice were sacrificed and lungs were removed and fixed in Bouin's fixative.
  • the first bar depicts the number of nodules observed in lungs of mice injected with cells cultured in normal conditions (20% O 2 ).
  • the second bar depicts the number of nodules observed in lungs of mice injected with cells cultured in normal conditions (20% O 2 ) and administered 2 x 10 "n M GTN.
  • the third bar depicts the number of nodules observed in lungs of mice injected with cells cultured in hypoxic conditions (1% O 2 ).
  • the fourth bar depicts the number of nodules observed in lungs of mice injected with cells cultured in hypoxic conditions (1% O 2 ) and administered 2 x 10" M GTN.
  • FIG. 3 shows circulating prostate specific antigen (PSA) levels in two patients, Patient A ( Figure 3 A) and Patient B ( Figure 3B) who had undergone radical prostectomy.
  • PSA prostate specific antigen
  • Figure 5 illustrates the effect of GTN and SNP on the hypoxic upregulation of in vitro invasion by MDA-MB-231 cells.
  • NO-mimetic drugs were added to the cells at the beginning of the 24-h assay and the invasion index for each treatment was calculated after counting the cells that penetrated through the membrane. Bars represent the mean, normalized, invasion indices ⁇ standard deviations. The value indicated by an asterisk
  • FIG. 6 illustrates urokinase plasminogen activator receptor (uPAR) expression in MDA-MB-231 breast cancer cells. Western blot analysis of uPAR protein expression by cells incubated in 1% O 2 in the presence of various concentrations of glyceryl trinitrate
  • GTN GTN
  • Figure 8 illustrates the effect of sGC disruption on the NO-mediated inhibition of uPAR expression in MDA-MB-231 breast carcinoma cells.
  • Figure 9 illustrates the effect of 8-Br-cGMP on hypoxia- and DFO-induced uPAR expression in MDA-MB-231 breast carcinoma cells.
  • Figure 10 illustrates the effect of PKG inhibition on uPAR expression in MDA- MB-231 breast carcinoma cells.
  • Figure 11 illustrates the effect of 8-Br-cGMP on the hypoxic upregulation of the in vitro invasiveness of MDA-MB-231 breast carcinoma cells.
  • Cells were allowed to invade through a reconstituted extracellular matrix for 24-h in 20%> O 2 or 0.5% > O 2 in the absence or presence of varying concentrations of 8-Br-cGMP.
  • the invasion index for each treatment was calculated after counting the cells that penetrated through the membrane. Values are presented as the relative mean of the invasion indices + standard deviation.
  • Figure 12 illustrates the effect doxorubicin (12.5-100 ⁇ M) on survival of human PC-3 prostate cancer cells. Cells were pre-exposed to 20% or 0.5% O 2 for 24 h before a 1 hr treatment with doxorubicin. Survival was assessed by clonogenic ability.
  • FIG. 13 illustrates the effect of a single dose of GTN on the hypoxia-induced resistance to doxorubicin.
  • Single asterisk (*) indicates a statistically significant difference (P ⁇ 0.0001) in survival compared with cells incubated in 20% O 2 at 12.5 ⁇ M doxorubicin.
  • Double asterisk (**) indicates a statistically significant difference (P ⁇ 0.0001) in survival compared with cells incubated in 0.5% O 2 alone.
  • Figure 14 illustrates the effect of MDA-MB-231 human breast carcinoma cell spheroid culture on resistance to doxorubicin over time. Following initial cell plating, spheroids formed for 24, 48, 72, 96 or 120h. At each time point, spheroids were exposed to 200 ⁇ M of doxorubicin, then disaggregated and plated for assessment of colony formation (survival). At this concentration of doxorubicin, survival in monolayer culture was zero (not shown). [53] Figure 15 illustrates the effect of 200 ⁇ M doxorubicin in combination with the
  • FIG. 16 illustrates the effect of GTN therapy on a patient with prostate cancer wherein the prostate is still intact. PSA levels in one patient with prostate cancer wherein the prostate is still intact are shown. This patient was administered three episodes of treatment, for approximately one month each, of GTN, 0.03 mg/hour, 24 hours a day.
  • FIG. 17 illustrates the effect of GTN therapy in combination with radiation therapy. Circulating PSA levels in one patient with prostate cancer wherein the prostate is still intact are shown. This patient was administered GTN chronically, transdermally at a concentration of 0.03 mg/hour. Two months after chronic GTN therapy was begun, the patient was administered radiation therapy. As shown, this combination therapy accelerated the rate of PSA decrease to within three months. The expected average for a similar decrease in PSA levels following radiation therapy alone is twelve months.
  • 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.
  • nitric oxide mimetic e.g., low dose
  • the methods and formulations of the present invention provide new therapeutic approaches for the treatment and prevention of cancer in animals.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ -fetoprotein (AFP)and ⁇ hCG for testicular cancer, ⁇ -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
  • 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).
  • PROXY-1 Protein Regulated by OXYgen-1
  • PAI-1 plasminogen activator inhibitor
  • uPAR urokinase-type plasminogen activator receptor
  • VEGF vascular endothelial growth factor
  • additional tumor markers to those exemplified herein can also be monitored in the present invention.
  • the tumor marker is detectable in a biological fluid such a serum, plasma or urine.
  • 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.
  • 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.
  • the known untoward effects of NO mimetics in animals without a malignant cell phenotype, cell, tumor and/or disease do not occur.
  • the nitric oxide mimetic increases, restores or maintains activity both in and around the cell (i.e.
  • 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.
  • Short-term nitric oxide mimetic therapy is generally administered at levels which increase nitric oxide mimetic activity of cells above normal physiologic levels.
  • 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.
  • administration of nitric oxide or compounds which deliver nitric oxide to human beings at doses conventionally employed to treat cardiovascular conditions i.e.
  • 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.
  • nitric oxide mimetics sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) sodium nitroprusside (SNP) and glyceryl trinitrate (GTN)
  • SNP sodium nitroprusside
  • GTN glyceryl trinitrate
  • 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.
  • nitric oxide mimetics 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.
  • inhibiting and preventing 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • malignant cell phenotype it is meant to encompass increases in metastasis, resistance to antimalignant therapeutic modalities, and angiogenesis.
  • 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.
  • conditions in this spectrum include, but are not limited to, benign prostatie hyperplasia and molar pregnancy.
  • 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.
  • 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”.
  • 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 -(l- iminoethyl)lysine) (L-NIL), L-N 5 -(l -iminoethyl)ornithine (L ⁇ - ⁇ IO), N°-methyl-L- arginine (L- ⁇ MMA), and S-nitrosoglutathione (S ⁇ OG);
  • GTN nitroglycerin
  • ISMN isosorbide 5-mononitrate
  • 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-[l- (3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl-l,3-propanediamine).
  • SSDD S,S-dinitrosodithiol
  • SNAP sodium nitroprusside
  • SNP S-nitroso-N- acetylpenicil
  • 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.
  • 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.
  • compounds which directly activate either particulate or soluble guanylyl cyclase such as natriuretic peptides (ANP, BNP, and CNP), 3-(5'-hydroxymethyl-2'furyl)-l- 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.
  • 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.
  • 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.
  • more than one NO mimetic is administered.
  • the NO mimetics target or act upon different parts of the NO pathway of the cell.
  • 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 (PDF) inhibitors useful as NO mimetics are those inhibiting PDE-1 through PDE-11.
  • 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.
  • 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). NO donors
  • 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.
  • 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), pentaerytliritol tetranitrate (PETN), ethylene glycol dinitrate, isopropyl nitrate, glyceryl-1-mononitrate, glyceryl-1,2- dinitrate, glyceryl-l,3-dinitrate, butane-l,2,4-triol trinitrate, and the like.
  • NTG nitroglycerin
  • ISDN isosorbide dinitrate
  • ISMN isosorbide mononitrate
  • ISMN isosorbide mononitrate
  • IS2N isosorbide-2-mononitrate
  • IS5N
  • 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-mo holinosydnonimine (SIN-1), ( ⁇ )-S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), (+)-(E)-ethyl-2-[(E)-hydiOxyimino]-5-nitro-3- hexeneamide (FK409), ( ⁇ )-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-5-nitro-3-hexen- 1
  • the compounds and methods of the present invention are not limited to the foregoing traditional nitric oxide mimetics.
  • these nitric oxide mimetics compounds include, for example, calcium channel blockers, ⁇ -adrenergic receptor antagonists and ⁇ -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.
  • 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
  • PDE inhibitors can be used at various doses such as high and low doses.
  • 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 + channel blockers are also available. -adrenergic receptor antagonists and ⁇ -adrenergic receptor agonists
  • Additional preferred compounds for use in the context of the present invention include, e.g., -adrenergic receptor antagonists and ⁇ -adrenergic receptor agonists.
  • Suitable ⁇ -adrenergic receptor antagonists include, for example, i -adrenergic receptor antagonists, ⁇ -adrenergic receptor antagonists and other nonspecific ⁇ -adrenergic ' receptor antagonists.
  • Preferred i -adrenergic receptor antagonists include, but are not limited to, prazosin, doxazosin, phenoxybenzamine, phentolamine, terazosin, tolazoline, etc., and are described in Goodman and Gilman, "Ebe Pharmaceutical Basis of Therapeutics," 9th Edition, Hardman, et al. (ed.), McGraw-Hill (1996).
  • Suitable ⁇ 2 - adrenergic receptor antagonists include, but are not limited to, yohimbine and are also described in Goodman and Gilman, "Tlie Pharmaceutical Basis of Therapeutics," 9th Edition, Hardman, et al. (ed.), McGraw-Hill (1996).
  • ⁇ 2 - adrenergic antagonists include, for example, post-synaptic ⁇ 2 -adrenergic antagonists.
  • post-synaptic ⁇ 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-l-yl)ethyl]-4,4-dimethyl-l,3- (2H,4H)-isoquinolindone dihydrochloride.
  • Suitable post-synaptic ⁇ 2 -adrenergic antagonists include, but are not limited to, idazoxan, rauwolscine, efaroxan, mianserin, and mirtazapine. Of these, mianserin and mirtazapine are particularly preferred.
  • Suitable ⁇ -adrenergic receptor agonists for use with the methods of the present invention include, but are not limited to, ⁇ .-adrenergic receptor agonists, ⁇ 2 -adrenergic receptor agonists, ⁇ 3 -adrenergic receptor agonists and other nonspecific ⁇ -adrenergic receptor agonists.
  • the ⁇ -adrenergic receptor agonist is a ⁇ 2 -adrenergic receptor agonist or a ⁇ 3 -adrenergic receptor agonists.
  • ⁇ -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).
  • Phosphodiesterase inhibitors Phosphodiesterase inhibitors
  • the compound is a phosphodiesterase inhibitor.
  • Cyclic nucleotide second messengers cAMP and cGMP
  • PDE cyclic nucleotide phosphodiesterases
  • Inhibitors of phosphodiesterases 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/IN inhibitors or PDE II/IN inhibitors). Below is a table setting forth various PDE inhibitors that are useful in the methods of the present invention.
  • the PDE inhibitor is a PDE N inhibitor.
  • PDE N 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-l,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)-l- benzyl thieno (3,2-c)pyrazole] for example.
  • the composition contains a phosphodiesterase type II (PDE II) inhibitor such as, e.g. , EH ⁇ A.
  • PDE II phosphodiesterase type II
  • the composition contains a phosphodiesterase type IN (PDE IN) inhibitor.
  • PDE IN phosphodiesterase type IN
  • Suitable phosphodiesterase type IN inhibitors include, but are not limited to, roflumilast, ariflo (SB207499), RP73401, CDP840, rolipram, mesopram, denbufylline, EMD 95832/3, cilomilast, RO-20-1724, and LAS31025.
  • the phosphodiesterase inhibitor is a dual selective phosphodiesterase inhibitor such as, e.g., a
  • PDE III/IN inhibitor e.g., zardaverine
  • phosphodiesterase inhibitors which can increase both cAMP and cGMP levels such as Satigrel (E5510, 4-cyano-5,5-bis(4- methoxyphenyl)-4-pentenoic acid).
  • the PDE inhibitor is an inhibitor of the PDE III isozyme, for example, Olprinone.
  • the PDE inhibitor is an inhibitor of the PDE IN isozyme family, or cAMP-specific and rolipram sensitive PDEs, which preferentially hydrolyze cAMP.
  • 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.
  • cAMP-dependent protein kinase activators [76]
  • the compound used to treat the disorders described herein is a cAMP-dependent protein kinase activator.
  • cAMP-dependent 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.
  • 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.
  • 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.
  • 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)).
  • SOD exogenous Mn or Cu/Zn superoxide dismutase
  • MnTBAP Mn(III) tetra(4-benzoic acid) porphyrin chloride
  • M40403 see, e.g., Salvemini, et al, Science, 286(5438):304- 306 (1999)
  • the present invention provides pharmaceutical compositions comprising a potassium channel activator.
  • 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).
  • the potassium channel activator is a
  • Maxi-K channel activator examples include, but are not limited to, estrogen-like compounds, such as estradiol (see, Nalverde, et al, SCIENCE, 285:1929-1931).
  • Benzodiazepines [79]
  • 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).
  • Adrenergic Nerve Inhibitors include, but are not limited to, alprazolam, brotizolam, chlordiazepoxide, clobazam, clonazepam, chlorazepate, demoxepam, diazepam, estazolam, flumazenil, flurazepam, hala
  • 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, ⁇ -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 ⁇ -adrenergic receptor antagonists.
  • Examples of sympathetic nerve terminal destroyers include, but are not limited to, ⁇ 2 -adrenergic receptor antagonists.
  • the compounds of the present invention are antidiarrheal agents.
  • suitable antidiarrheal agents include, but are not limited to, diphenoxylate, loperamide, bismuth subsalicylate, octreotide, etc. (see, e.g., Goodman and Gilman, supra).
  • the compounds of the present invention are HMG-CoA reductase inhibitors.
  • 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). Smooth Muscle Relaxants
  • the compounds of the present invention are smooth muscle relaxants such as, e.g., hydralazine, papaverine, tiropramide, cyclandelate, isoxsuprine and nylidrin.
  • smooth muscle relaxants such as, e.g., hydralazine, papaverine, tiropramide, cyclandelate, isoxsuprine and nylidrin.
  • 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.
  • the adenosine receptor modulators are combined with at least one other muscle-relaxing agent.
  • the compounds of the present invention are adenosine receptor modulators such as methylxanthines.
  • adenosine receptor modulators include theophylline and dyphylline.
  • Preferred agents are selected from those described with reference to the compositions of single agents or combinations above.
  • 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.
  • a related compound i.e., dyphylline
  • 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.
  • compositions comprising adenylyl cyclase activators, either alone or in combination with other compounds or agents described herein.
  • the adenylyl cyclase activator forskolin is preferred.
  • adenylyl cyclase activators include, but are not limited to, N6, 02 - 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.
  • the present invention provides compositions comprising endothelin receptor antagonist, either alone or in combination with other compounds disclosed herein.
  • 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
  • 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.
  • cGMP-dependent Protein Kinase Activators [90] 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.
  • cGMP mimetics include, but are not limited to, 8-bromo-cGMP, dibutyryl-cGMP, Rp-cGMPS, and Sp-cGMPS
  • useful dual activators include compounds such as, e.g., Sp-8-pCPT- cGMPS, Sp-8-bromo-cGMPS and 8-CPT-cAMP.
  • Guanylyl Cylase Activators 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.
  • 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.
  • 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.
  • 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 (J K) and exocytosis, resulting in the release or elaboration of inflammatory mediators.
  • SOC inhibitors include for example statins in the ⁇ -lactone form such as lovastatin, mevastatin, fluvastatin, pravastatin, dalvastatin, cerivastatin, atrovastatin and simvastatin.
  • 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.
  • animal it is meant to include all mammals, and in particular humans.
  • 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.
  • 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.
  • uPAR is a high affinity cell surface receptor for pro-urokinase-type plasminogen activator (pro-uPA).
  • pro-uPA pro-urokinase-type plasminogen activator
  • 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).
  • ECM extracellular matrix
  • 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.
  • hypoxia-induced uPAR up-regulation has been suggested (Graham et al. Int. J. Cancer 1999 80:617-623).
  • 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.
  • NOS NO synthase
  • the promotor region of uPAR contains binding sites for transcription factors such as activator protein-1 (AP-1), Sp-1/3 and nuclear factor KB (NFKB). Hypoxia Inducible Factor 1 (HIF-1) levels may also contribute to the transcriptional activation of the uPAR gene, as previous examination of the sequences 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 ⁇ M) of NO mimetics such as SNP, S-nitroso-L-glutathione and 3-morpholinosydnonimine.
  • NO mimetics such as SNP, S-nitroso-L-glutathione and 3-morpholinosydnonimine.
  • 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. [102] 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.
  • NF- ⁇ B as a molecular target for developing anti- cancer therapy has been reviewed extensively by several investigators.
  • Various known NFKB inhibitors such as NSAIDs, glucocorticoids, COX 2 inhibitors, and more recently proteasome inhibitors (blocks NF- ⁇ B activation), have been shown to be effective or potentially effective cancer treatment options.
  • Known NFKB 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.
  • NO may function as the feedback inhibitor of NFKB upregulation activation.
  • 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.
  • 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.
  • anti- androgens e.g. flutamide
  • anti-estrogen e.g. tamoxifen
  • 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.
  • Endogenous androgens including testosterone and 5- ⁇ -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.
  • the majority of the biological activities of testosterone and DHT are elicited by the cytosolic androgen receptor (AR).
  • AR cytosolic androgen receptor
  • 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.
  • 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.
  • 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.
  • NO mimetics can be used with chemo-and or radio- therapeutic treatment to ensure eradication of cancers during the treatment phase, and can be used as stand-alone therapy during the remission. If low dose of chemotherapy is employed to prevent cancer recurrence, NO mimetics can be used with the low dose chemotherapy to prevent or prolong the time to cancer recurrence and eventually, prolong the survival time of cancer patients.
  • the addition of NO mimetic therapy could also 1) reduce the dependence on narcotic pain relief agents and thus associated adverse events due to their use and yet enhance the effectiveness of these agents, 2) prevent progressive lost of bone mineral density and thus, the development of osteoporosis and the risk of bone fracture, and 3) improve overall quality of life of cancer suffers.
  • nitric oxide is a primary mediator of cellular adaptive responses to changes in oxygen levels in and around the cell.
  • nitric oxide mimetic activity can be increased, restored or maintained at a level which inhibits or prevents a malignant cell phenotype.
  • the effect of maintaining low oxygen levels on cells was limited to inhibiting basal levels of endogenous nitric oxide production.
  • cancer cell lines acquire one or more of the following malignant cell phenotypic properties: they increase their lung-colonization ability following i.v.
  • nitric oxide mimetics SNP and/or glyceryl trinitrate (GTN) inhibit the hypoxic up-regulation of uPAR and PAI-1, as well as PROXY-1.
  • GTN glyceryl trinitrate
  • Similar low dose nitric oxide mimetic therapy is expected to also be effective in inhibiting hyponitroxic upregulation of these genes such as that observed in cells treated with L-NMMA (0.5 mM), i.e. inhibiting and preventing a malignant cell phenotype.
  • PROXY-1 mRNA levels were very low in cells cultured in 20%> O 2 .
  • levels of PROXY-1 mRNA were increased in cells cultured in l%o O 2 which were untreated or treated with 10 "7 M GTN.
  • levels of PROXY-1 were much lower in hypoxic cells treated with a low dose, 10 "11 M GTN.
  • the effects of the nitric oxide mimetics SNP and GTN on PAI-1 mRNA levels in breast cancer cells were examined.
  • hypoxic cells with low doses 10 "11 M GTN and 10 "12 M SNP, of a nitric oxide mimetic resulted in a decrease in gelatinases secreted from the cells as compared to untreated hypoxic cells.
  • 10 "11 M GTN and 10 "12 M SNP 10 "11 M GTN and 10 "12 M SNP
  • inhibition of the hypoxic up-regulation of these genes was then shown to result in a decrease in cellular invasiveness and drug resistance.
  • the invasive ability of cells in hypoxic conditions in the presence or absence of nitric oxide mimetics was also assessed using Matrigel invasion chambers (modified Boyden chambers). In these in vitro invasion assays, either breast cancer cells (see Figure 1) or HTR-8/SVneo invasive trophoblasts were plated on Matrigel-coated membranes.
  • hypoxic breast cancer cells were then incubated under hypoxic or normal conditions, alone or in the presence of nitric oxide mimetics.
  • the invasion index for each treatment was determined by staining the cells which invaded through the membrane and counting them.
  • treatment with low doses of nitric oxide mimetics significantly reduced hypoxic cell invasiveness as compared to untreated hypoxic cells.
  • the invasive indices of hypoxic breast cancer cells treated with 10 "10 M SNP and 10 "11 M GTN were similar to or even lower than cells cultured under non-hypoxic conditions.
  • mice were administered via tail vein injection a bolus of metastatic melanoma cells. Immediately after injection the mice were divided into two groups. The first group was placed in a chamber with a continuous flow of a gas mixture comprising 21% O 2 (room air).
  • mice were injected with mouse melanoma cells which were pre-incubated for 12 hours in 1 %> or 20% O 2 in the presence or absence of a low concentration of a nitric oxide mimetic (GTN; 2 x 10 "n M).
  • GTN nitric oxide mimetic
  • mice After fourteen days, the mice were sacrificed and the lungs were visually observed for metastatic nodules. In addition, the number of lung nodules in these mice was compared. Lungs of animals that had been administered hypoxic and non-hypoxic melanoma cells treated with the nitric oxide mimetic prior to injection exhibited statistically less metastatic nodules as compared to animals administered either untreated hypoxic and non-hypoxic melanoma cells (see Figure 2). Specifically, the in vitro pre-treatment with 2xlO " ⁇ M GTN decreased the hypoxia-stimulated lung nodule formation by 85% and, even in 20% oxygen, the GTN pre-treatment reduced the extent of metastasis by more than 60%.
  • Characterization of the lung nodule frequency by fertile revealed a consistent pattern of suppression throughout the NO-mimetic treated group compared to the L-NMMA treated group.
  • the metastasis in the GTN-treated group was significantly below even the control levels in the highest tertile.
  • these data indicate that the levels of NO, and not oxygen itself, determine the severity of the metastatic phenotype.
  • the effect of low-concentration GTN treatment on lung nodule formation was not due to a non-specific cytotoxic or growth inhibitory effect on the cells as they had similar in vitro colony-forming ability as untreated cells.
  • results from the studies in this murine model can be used to predict drug disposition in other species including humans, to define pharmacokinetic equivalence in various species including humans and design dosing regimes for other experimental animal models and for human clinical studies.
  • Such pharmacokinetic scaling is performed routinely based upon data such as provided herein as evidenced by references such as Mordenti, J. J. Pharm. Sci. 1986 75(11):1028-1040.
  • the ability of an NO mimetic to reduce disease progression in humans was demonstrated.
  • nitric oxide mimetic treated hypoxic cells were comparable to those observed in untreated non-hypoxic cells and non-hypoxic cells treated with the nitric oxide mimetic. These results have been confirmed in multiple human cancers as well as mouse cancers and with other antimalignant therapeutic modalities. [125] Thus, these studies demonstrate that a malignant cell phenotype such as that induced by hypoxia can be inhibited and prevented by increasing (restoring) the level of nitric oxide mimetic activity.
  • nitric oxide mimetic activity so as to induce a malignant cell phenotype
  • factors known to lower cellular nitric oxide mimetic activity so as to induce a malignant cell phenotype include, but are not limited to, decreases in arginine levels, exposure to endogenous nitric oxide synthase antagonists such as L-NMMA and ADMA, exposure to endogenous nitric oxide scavengers such as superoxide, changes in nitric oxide synthase expression, changes in cofactors such as GSH and NADPH, glucose deprivation, surgical procedures, administration of anesthetic agents, administration of pharmacologic agents which alter circulation such as, but not limited to antihypertensive agents, and traumatic injuries including, but not limited to those associated with blood loss, decreased blood volume, and hemorrhage.
  • the present invention relates to methods of inhibiting and preventing a malignant cell phenotype resulting from these and other factors by administering low doses of one or more
  • the low dose nitric oxide mimetic therapy of the present invention will also prevent the malignant cell phenotype of vascular endothelial cells which ultimately results in recruitment of such cells by a tumor and development of a blood supply to the tumor, also known as angiogenesis.
  • Attempts at cutting off tumor blood supply by blocking VEGF in vascular endothelial cells have been relatively unsuccessful as cancer treatments.
  • the presence of VEGF in tumors has been shown to suppress tumor invasiveness.
  • agents which remove or block the actions of VEGF such as anti-VEGF antibody, actually generate more aggressive cancer cell phenotypes.
  • Formulations for the administration of nitric oxide mimetics in accordance with the method of the present invention can take the form of ointments, transdermal patches, transbuccal patches, injectables, nasal inhalant forms, spray forms for deep lung delivery through the mouth, orally administered ingestible tablets and capsules, and tablets or lozenges, or "lollipop" formulations for administration through the oral mucosal tissue.
  • the latter formulations included tablets, lozenges and the like which are dissolved while being held on or under the tongue, or in the buccal pouch. It is preferred that the pharmaceutical preparations provide a low dose of the nitric oxide mimetic sufficient to increase, restore or maintain nitric oxide mimetic activity at a level which inhibits or prevents a malignant cell phenotype, also referred to herein as a therapeutically effective amount, during the period in which cellular nitric oxide mimetic activity of cells is lowered. Also preferred are formulations comprising more than one NO mimetic. In this embodiment, it is preferred that the NO mimetics target or act on different parts of the NO pathway.
  • 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.
  • a compound that inhibits cyclic nucleotide (e.g. cAMP or cGMP) degradation such as a phosphodiesterase inhibitor.
  • lung cancer including non-small cell lung cancer and small cell lung cancer; mesothelioma; thymic tumors; breast cancer, such as ductal carcinoma in situ, lobular carcinoma in situ, ductal carcinoma of special type, and invasive lobular carcinoma; colorectal cancer; anal cancer; esophageal cancer; gastric cancer; small intestine and carcinoid tumors; cancers of the liver such as primary liver cancer and hepatocellular carcinoma; cancer of the biliary tract, including cholangiocarcinomas; tumors of the gall bladder; pancreatic cancer; endocrine cancers such as thyroid cancer, adrenal cancer, and neuroblastoma; genitourinary cancers such as renal cancer, Wilms' tumor, cancer of the bladder and ureter, prostate cancer, and testicular cancer; gynecological cancers such as ovarian cancer, cancer of the uterine
  • Lung cancers are grouped as Surgical removal of non-small cell bronchogenic non-small cell (NSCLC) or carcinoma continues to offer best possibility of a small cell (SCLC), but cure. within the former certain
  • Radical radiotherapy is indicated for patients with patterns of disease do relate who are unfit for surgery, or have good to histological subtype.
  • squamous cancers encompassed in a radical volume.
  • the standard typically arise in proximal international dose is 60 Gy in 30 fractions over 6 segmental bronchi and grow weeks.
  • the standard treatment volume in most of slowly, disseminating the world is the primary tumor and hilar and relatively late in their course. mediastinal lymph nodes, with a 1-2 cm margin.
  • Adenocarcinomas are often Chermotherapy delivered synchronously with peripheral in origin and even radiotherapy is being investigated.
  • small respectable lesions carry a risk of occult Cisplatin-based therapy significantly improves metastases. survival and quality of life.
  • the most studied regimen is MIC (mitomycin C, isosfamide, and cisplatin).
  • SCLC small cell lung cancer
  • TI thoracic irradiation
  • PCI prophylactic cranial irradiation
  • CAV cyclophosphamide, doxorubicin and vincristine
  • EP etoposide and cisplatin
  • MPM MPM mesothelioma
  • Thymic Tumors Tumors derived from the The contemporary standard of treatment for thymus (thymomas) thymoma is radical surgery whenever possible.
  • Type of Cancer Description of the Cancer Standard of Care and Investigational Treatments comprise approximately 20% Total resection alone is adequate therapy for early of all mediastinal tumors and stage thymoma.
  • Adjuvant therapy should be used are the most common tumor for local disease with demonstration or suspicion in the anderior mediastinum. of invasion.
  • radiotherapy is recommended in but are rare before the age of doses of 50-60 Gy given in 20-30 20 and peak between 40-60 fractions.Locally advanced, relapsing, or years. disseminated disease should be treated with combination chemotherapy including
  • Cisplatin-based regimens are growing 'low-grade' recommended.
  • malignant tumors It is believed that they derive from epithelial elements, but the tumors retain the capacity for production of T cells.
  • Breast Cancer Breast cancer is the most Early breast cancer is defined as disease that can common solid cancer be completely extirpated by surgery. The occurring in women. It is management of this disease comprises: treatment estimated that 5-10% of of the breast and axilla; pathological staging to female breast cancer is due to direct adjuvant therapy; adjuvant therapy- inheritance of a mutated endocrine, chemotherapy, radiotherapy; and copy of either BRCA 1 or follow-up. BRCA2. Other genes contibute less frequently to All patients require removal of the primary tumor familial breast cancer. Male with either wide local excision or mastectomy.
  • breast cancer is rare (0.7% of Breast irradiation has been shown to reduce the all male cancers) with a peak risk of local recurrence after breast-conserving incidence 10 years later than surgery.
  • the whole breast is treated women. It may occur in with tangential fields to a dose of 50 Gy in 25 association with Klinefelter's fractions (or an equivalent dose-fractionation syndrome. regimen), with care taken to minimize the volume of lung and heart irradiated.
  • DCIS Ductal carcinoma in situ
  • Active agents include type antracyclines, alkylating agents, and antimetabolites. Combinations such as FAC (5-
  • a number of pathological fluorouracil, doxorubicin, cyclophosphamide) variants are identified, produce response rates of 40-60%, with a median namely medullary time to progression of around 8 months. 20-50% carcinoma, tubular of women respond to second-line chemotherapy carcinoma, and mucinous with a taxane and studies are currently evaluating carcinoma. the promising combination of anthracycline plus
  • Invasive lobular carcinoma taxane as first-line chemotherapy.
  • Low-dose radiotherapy provides palliation in these patients.
  • Colorectal cancer Colorectal cancer is the Surgery is the mainstay of curative therapy for fourth commonest cancer colorectal cancer. Curative resection requires the worldwide. Around two- excision of the primary tumor and its lymphatic thirds of a million people drainage with an enveloping margin of normal will present with the disease tissue. each year. It affects men and
  • the aim of adjuvant chemotherapy is to eradicate women almost equally and micro-metastases and thereby prevent future tends to be more common in relapse.
  • Current available chemotherapy does not 'developed' countries and is completely eradicate bulky, advanced metastatic particularly common in the bowel cancer. It does however eradicate mico- US, Europe and Australia. metastases in a proportion of patients.
  • the current 'international standard' adjuvant almost always chemotherapy regimen for colon carcinoma is 5- andenocarcinomas.
  • the fluorouracil (5FU) given in combination with tumor often starts as a folinic acid, by bolus intravenous injection. polypoidal mass and then tends to infiltrate into and through the bowel wall.
  • Oxaliplatin (which forms covalently bonded adducts with DNA).
  • Raltitrexed is a potent inhibitor of thymidylate synthase. It can be used as an alternative to 5FU- based regimes for first-line treatment. It does not require coadministration of a biochemical modulator.Intra-hepatic 5FU, following surgical placement of a hepatic artery catheter, may be considered for patients with unresectable liver metastases.
  • Radiotherapy in colonic cancer is limited to the palliative situation in most circumstances.
  • the rectum is a suitable target for radiotherapy.
  • Radiotherapy has been used in both the pre- operative and post-operative settings in this disease.
  • In the pre-operative situation there are a group of patients who present with large fixed or tethered tumors that are non-resectable.
  • the conversion rate to respectability is 35-75%, with a dose of 50-60 Gy given over a five-week period. This group is also offered combined chemo- irradiation
  • Anal cancer Most anal tumors arise from The standard treatment for anal canal tumors was the epidermal elements of the abdominoperineal resection, while anal margin anal canal lining (squamous growths were viewed as equivalent to skin tumors cell- 85% of anal tumors), elsewhere and treated by local excision.
  • radiotherapy and/or chemotherapy glandular mucosa of the have become increasingly popular and in many uppermost part of the anal cases the treatment of choice. canal or from the anal ducts and glands
  • Non-surgical treatment (chemo-irradiation) for anal cancer has become increasingly popular.
  • the (adenocarcinomas). drugs used are usually 5FU and mitomycin C.
  • the Malignant melanoma of the anus is very rare .
  • radiotherapy consists of 30 Gy of external-beam irradiation over a period of three weeks. Traditionally, the anal region is divided into the anal canal
  • Adenocarcinoma is radiosensitive, it is and the anal margin or verge. usually treated by radical surgery.
  • Esophageal cancer Surgical resection is the treatment of choice for early stage disease.Advanced disease is
  • Squamous cell carcinoma is chemosensitive Post-resection irradiation the most common improves the loco-regional control in patients with histological type, but positive resection margins, but not if there is nodal adenocarcinoma is increasing involvement.
  • the dose is limited to 50 Gy in 25 Type of Cancer Description of the Cancer Standard of Care and Investigational Treatments
  • HCC Hepatitis C infection
  • Tumors may also be including doxorubicin, mitozantrone, methotrexate found incidentally at the time and cisplatinum have been used as single agents or of post-mortem examination in combination with 5FU. in patients with cirrhosis.
  • Surgical ligation of the hepatic artery and insertion of a hepatic artery catherter have been used to deprive the tumor of oxygenated blood and to provide a route for the direct administration of cytotoxic chemotherapy.
  • Such techniques have been superceded by the use of chemo- embolization via radiologically placed catheters in the hepatic artery. This technique is less invasive and, using chemotherapy such as adriamycin combined with lipiodol uptake by the tumor, is demonstratable on CT scan. Repeat embolization is performed at 2-3 month intervals and tumor regression with associated regeneration of normal liver may render some tumors resectable.
  • tumors of Chonangiocarcinomas should be resected if there tract the biliary tract are are no distant metastases and no irreparable adenocarcinomas of the involvement of the hepatic artery and portal vein. papillary, nodular, or It is advisable to perform a full lymph-node sclerosing type. Papillary dissection. tumors develop more
  • cholangiocarcinomas are not generally commonly in the gall bladder considered chemosensitive or radiosensitive, or in the distal bile duct; responses to chemotherapy with cisplatin and 5FU sclerosing and nodular and folinic acid have been observed. Newer agents tumors, in the proximal bile such as gemcitabine and irinotecan are under duct. Adenocarcinomas investigation. without specific features are the most common. For tumors of the gall bladder, incidental tumors diagnosed after a laparoscopic cholecystectomy
  • Tumors of the bile duct can need no further action unless the gall bladder was develop within the bead of ruptured. Tumors of more advanced stages should the pancreas , in the be treated with radical surgery. Post-operative hepatoduodenal ligament , or chemotherapy with cisplatin has yet to be proven at the level of the hepatic to provide beneficial results. Radiotherapy is used hilum,. Hilar in cases of incomplete resection. cholangiocarcinomas (Klatskin's tumors) account for more than half in most series.
  • Cholangiocarcinomas especially of the sclerosing and nodular type, infiltrate along the walls of the ducts and the perineural tissue before obstructing the lumen. Metastases to the lymph nodes are seen in some patients undergoing surgery. Direct duodenal invasion and peritoneal carcinomatisis Type of Cancer Description of the Cancer Standard of Care and Investigational Treatments
  • PSA Prostate cancer Pathogenesis of cancer of the Measurement of PSA should be done in all prostate gland is clearly patients with outflow symptoms, and is routinely androgen-dependent. Since used as a screening tool in the US. PSA levels >4 the development of assays to mg/ml are considered to be suspicious; transrectal measure prostate-specific ultrasound and needle biopsy are called for in antigen (PSA) to diagnose these patients. PSA levels >50 ng/ml may be prostatie disease, the indicative cf distant metastases. condition has been
  • Treatments used to produce cessation of androgen-driven growth of the cancer include surgical castration, estrogens, steroidal anti-androgens such as cyproterone acetate, non-steroidal anti-androgens (flutamide, bicalutamide), and medical castration with LHRH agonists.
  • the combination of medical castration and anti-androgen therapy has been used.
  • chemotherapeutic anthracyclines and the related mitoxantrone have been reported to be useful. Radiation therapy offers useful palliation of advanced disease both in bone and soft tissue. In addition, radioactive strontium given by IV injection (150 MBq) has proved effective in relieving bone pain and delaying the progression of symptomatic bone disease.
  • Testicular cancer Testicular cancer is the The management of seminoma and non- commonest cancer in young seminomatous germ cell tumors depends on the men. The majority of stage of the disease. Initial management of testicular cancers are germ testicular germ cell tumors generally includes cell tumors which arise in the inguinal orchidectomy, although in cases with germinal epithelium. Both widespread metastases immediate chemotherapy seminomas and non- may be appropriate. Carcinoma in situ will seminomatous germ cell progress to invasive cancer, either seminoma or tumors are thought to arise non-seminomatous germ cell tumors. Low-dose from pre-existing carcinomas ratiotherapy to the testis (20 cGy in 10 fractions in situ. Non-germ cell over 2 weeks) is useful in early stage patients. testicular tumors have been Standard doses of radiotherapy (30 Gy in 15 observed as well.. fractions over 3 weeks) and chemotherapy are appropriate for later stage patients.
  • Non-seminomatous germ cell tumors produce markers in the form of the human chorioric gonadatrophin (HCG) and/or alpha feta protein (AFP) in a majority of cases. Seminomas have no reliable tumor marker with which to monitor disease, although HCG may be raised in about 25% of cases.
  • the principal active agents are cisplatin, mitomycin C, ifosfamide, methotrexate, 5-fluorouracil, and bleomycin. Modern management of advanced disease regularly involves the use of concurrent or sequential chemotherapy and radiotherapy.
  • vaginal and vulvar Most vaginal malignancies Radical radiotherapy with a combination of pelvic cancer are metastatic, from primary external-beam and utero-vaginal intra-cavitary sites in the cervix, vulva, brachytherapy is the treatment of choice for endometrium, or trophoblast vaginal cancer. Lower vaginal involvement should (choriocarcinoma). The most prompt consideration of either additional groin common histological types of node dissection or irradiation. primary cancer are squamous
  • Chemo-irradiation cancer the majority are (typically 5FU and mitomycin C combined with squamous carcinoma; other radiotherapy) used in cases of advanced disease types include basal shows encouraging results. carcinoma and malignant melanoma.
  • Trophoblastic tumors Gestational trophoblastic Treatment of hydatiform moles may include disease (GTD) includes a gentle suction curettage.
  • GTD disease
  • Hysterectomy or cesarean spectrum of disorders section increases the risks of chemotherapy being ranging from the pre- required to eradicate persistent trophoblastic malignant complete disease.
  • the stage of the disease is used to hydatidiform mole (CHM) determine what chemotherapy regimen is and partial hydatidiform appropriate.
  • Methotrexate is commonly used in mole (PHM), to the later stage patients.
  • Combination chemotherapy malignant invasive mole comprising etoposide, methotrexate, and gestational choriocarcinoma, actinomycin D (EMA) alternating weekly with and the highly malignant cyclophosphamide and cincristine (CO) may be placental-site trophoblastic used in very late stage patients.
  • PSTT Treatment with tumor
  • Both CHM methotrexate or EMA CO regimens continues and PHM can develop into until the HCG has been normal for six weeks. invasive moles. However, it
  • Choriocarcinoma where it can be safely achieved, is thought that only CHM excision biopsy of a metastasis should be used to may progress to the highly confirm the diagnosis. Patients are scored and malignant choriocarcinoma treated the same as for molar disease. and the rare PSTT.
  • Choriocarcinoma is a highly forms of GTD and it is relatively chemoresistant. malignant form that The best management is hysterectomy when the usually presents within one disease is localized to the uterus. When metastatic year of pregnancy. One third disease is present, patients can respond to multi- of patients present with agent chemotherapy either alone or in combination metastases to the liver, brain or lung. with surgery.
  • PSTT can develop following a term delivery, non-molar abortion, or CHM.
  • PSTTs are slow-growing malignant tumors composed mainly of cytotrophoblast with very little syncytiotrophoblast, so Type of Cancer Description of the Cancer Standard of Care and Investigational Treatments bone marrow.
  • Chlorambucil is first-line therapy.
  • Single-agent prednisolone (lmg/kg/day) is useful in advanced
  • B-cell chronic lymphocytic disease Combination chemotherapy using COP or leukemia is the common CHOP has been evaluated.
  • Purines are considered leukemia of late middle-age. the best second-line therapy.
  • Chlorambucil also used at a dose of 25mg m 2 IV for 5 days on a 4-6 week cycle for up to 6 cycles.
  • Radiotherapy is effective local treatment for lymph nodes. Splenectomy is effective for massive splenomegaly, anemia or thrombocytopenia and for conditions refactory to prednisolone and cytotoxic therapy.
  • Hodgkin's disease Standard therapy for early stage Hodgkin's disease comprises nodal irradiation. Chemotherapy combined with limited (involved field) radiotherapy has been investigated. Chemotherapy regimens include VBM (vinblastine, bleomycin, methotrexate) and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine). In cases with good prognosis involved field radiotherapy has been used alone successfully.
  • MOPP mustine, vincristine, procarbazine, prednisolone
  • Doxorubicin-based chemotherapy particularly ABVD
  • Newly introduced brief-duration regimens include: Stanford V (mustine, doxorubicin, vinblastine, prednisolone, vincristine, bleomycin, etoposide); BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisolone); and may be combined with limited field radiotherapy to sites of bulk disease.
  • Non-Hodgkin's For low grade NHL, treatment is reserved until lymphomas(NHL) lymphomas(NHL) are a active symptoms present.
  • Local radiotherapy is group of malignant diseases applied to bulky sites. Single-agent chemotherapy arising from cells of the (chlorambucil), as well as combination therapy immune system. (CHOP) are standard. New treatments include purine analogs (fludarabine, 2-CDA), autologous
  • High grade NHL is tranplant, alpha interferon, palliation with anti- considered to have a strong CD20 antibody (rituximab), antisense therapy to tendency to involve the CNS suppress Bcl-2 protein. and is comprised of Burkitt's lymphoma,lymphoblastic Burkitt's lymphoma is treated with intensive lymphoma, and diffuse large chemotherapy with methotrexate, B-cell NHL. Diffuse large B- cyclophosphamide, ifosamide, and with intrathecal cell NHL is the commonest therapy.
  • compositions of the present invention comprise a therapeutically effective amount of the nitric oxide mimetic formulated together with one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulf
  • Coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the formulation according to the judgment of the formulator.
  • the formulations of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), supralingually (on the tongue) sublingually (under the tongue), bucally (held in the buccal pouch), or as an oral or nasal spray.
  • the oral spray may be in the form of a powder or mist which is delivered to the deep lungs by oral inhalation.
  • the invention provides topical sustained and prolonged release pharmaceutical compositions comprising one or more pharmacological compounds described supra, and a pharmaceutically acceptable carrier, to treat a malignant cell phenotype disorders.
  • a topical formulation includes 75% (w/w) white petrolatum USP, 4% (w/w) paraffin wax USP/NF, lanolin 14% (w/w), 2% sorbitan sesquioleate NF, 4% propylene glycol USP, and 1% compound of the present invention.
  • Such compositions are useful in the treatment of cancer and in controlling and reducing pain associated therewith.
  • compositions may comprise a unit dosage of one or more particular active agent(s) (e.g., a NO donor, calcium channel blocker, cholinergic modulator, ⁇ -adrenergic receptor antagonist, ⁇ -adrenergic receptor agonist, phosphodiesterase inhibitor, cAMP-dependent protein kinase activator, superoxide scavenger, potassium channel activator, SOC inhibitors, benzodiazepine, adrenergic nerve inhibitor, antidiarrheal agent, HMG-CoA reductase inhibitor, smooth muscle relaxant, adenosine receptor modulator, adenylyl cyclase activator, cAMP mimetic, endothelin receptor antagonist, bisphosphonate, cGMP-dependent protein kinase activator, and cGMP mimetic.
  • active agent(s) e.g., a NO donor, calcium channel blocker, cholinergic modulator, ⁇ -adrenergic receptor antagonist
  • compositions are administered in unit dosage form to a subject in need of such treatment.
  • Topical sustained and prolonged release compositions are typically variants which include 1) an absorbent in a hydrophilic base; 2) an absorbent in a hydrophobic base; and 3) coated beads containing an absorbent matrix dispersed in a suitable vehicle.
  • methods of treating cancer comprising topically administering an effective amount of such compositions (e.g., in unit dosage form) to the appropriate area of the subject in need of such treatment.
  • Such hydrophilic compositions and preparations of the invention comprise a compound of the invention and a polymer, such as cellulose (methyl cellulose, ethyl cellulose, hydroxy propyl cellulose, etc.), higher molecular weight polyethylene glycol, methacrylic-acrylic acid emulsion, hydrogel, carbopol, ethyl vinyl acetate copolymer, or polyester, etc., to bind the compound of interest to the polymer.
  • the compound-polymer matrix is then dispersed in a hydrophilic vehicle to form a semi-solid.
  • the water in the semi-solid preparation is adsorbed and the polymer matrix with the active ingredient (i.e., the pharmaceutical compound) remains as a coating in the area to which it has been applied.
  • the pharmaceutical compound is then slowly released from this coating.
  • Hydrophobic compositions and preparations of the invention employ similar polymers as used in the hydrophilic preparations, but the polymer/compound matrix is dispersed into a vehicle, such a plastibase, in the hydrophobic compositions and preparations.
  • Plastibase is a mineral oil base that only partially dissolves the pharmaceutical compound.
  • the semi-solid composition forms a thin coating to which the composition has been applied such as, e.g., the vagina or urethral tract) and slowly releases the active compound. The prolonged action is controlled principally by the solubility of the active ingredient in the vehicle.
  • the present invention also provides coated beads which are produced by first absorbing a compound of the present invention, or a combination of compounds, on a cellulosic material blended with polyethylene glycol, filler, binder and other excipients. The resulting matrix is then extruded and spheronized (e.g. , the process of making into spheres) to create small beads. The beads are then coated to an appropriate thickness with one or more of a suitable material, such as a methacrylic-acrylic polymer, polyurethane, ethyl vinyl acetate copolymer, polyester, silastic, etc. The coating on the beads acts as a rate controlling membrane that regulates the release of the compound from the core beads.
  • a suitable material such as a methacrylic-acrylic polymer, polyurethane, ethyl vinyl acetate copolymer, polyester, silastic, etc.
  • the invention provides pharmaceutical compositions suitable for oral administration which are provided in unit dosage form comprising per unit dosage a NO donor, calcium channel blocker, cholinergic modulator, ⁇ -adrenergic receptor antagonist, ⁇ -adrenergic receptor agonist, phosphodiesterase inhibitor, cAMP- dependent protein kinase activator, superoxide scavenger, potassium channel activator,
  • SOC inhibitors benzodiazepine, adrenergic nerve inhibitor, antidiarrheal agent, HMG- CoA reductase inhibitor, smooth muscle relaxant, adenosine receptor modulator, adenylyl cyclase activator, cAMP mimetic, endothelin receptor antagonist, bisphosphonate, cGMP-dependent protein kinase activator, cGMP mimetic, and a pharmaceutically acceptable carrier.
  • Such compositions are useful for treating cancer, including those disorders and conditions described above.
  • an oral formulation such as a lozenge, tablet, or capsule is used.
  • Methods of manufacture of these formulations are known in the art, including but not limited to, the addition of a pharmacological agent to a pre-manufactured tablet; cold compression of an inert filler, a binder, and either a pharmacological compound or a substance containing the compound (as described in U.S. Patent No. 4,806,356); and encapsulation.
  • Another oral formulation is one that can be applied with an adhesive, such as the cellulose derivative, hydroxypropyl cellulose, to the oral mucosa, for example as described in U.S. Patent No. 4,940,587.
  • This buccal adhesive formulation when applied to the buccal mucosa, allows for controlled release of the pharmacological agent into the mouth and through the buccal mucosa.
  • the compounds of the present invention can be incorporated into these formulations as well.
  • aerosol For delivery to the nasal or bronchial membranes, typically an aerosol formulation is employed.
  • aerosol includes any gas-borne suspended phase of the pharmacological agent that is capable of being inhaled into the bronchioles or nasal passages.
  • aerosol includes a gas-borne suspension of droplets of the compounds of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of a pharmacological compound of interest suspended in air or other carrier gas, which may be delivered by insufflation from an inhaler device, for example.
  • the preferred range of concentration of the pharmacological agent is 0.1-100 mg/ml, more preferably 0.1-30 mg/ml, and most preferably, 1-10 mg/ml.
  • the solutions are buffered with a physiologically compatible buffer such as phosphate or bicarbonate.
  • a physiologically compatible buffer such as phosphate or bicarbonate.
  • the usual pH range is 5 to 9, preferably 6.5 to 7.8, and more preferably 7.0 to 7.6.
  • sodium chloride is added to adjust the osmolarity to the physiological range, preferably within 10% of isotonic.
  • Formulation of such solutions for creating aerosol inhalants is discussed in Remington, Pharmaceutical Sciences, see also, Ganderton, et al, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda, Critical Reviews in Therapeutic Drug Carrier Systems, 6:213-313 (1990); and Raeburn, et al, J. Pharmacol. Toxicol Methods, 27:143-159 (1992).
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and
  • the oral formulations can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P.
  • injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by inco ⁇ orating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the abso ⁇ tion of the nitric oxide mimetic from subcutaneous or intramuscular injection can be slowed. This can be accomplished by the use of a liquid suspension of crystalline or amo ⁇ hous material with poor water solubility. The rate of abso ⁇ tion of the nitric oxide mimetic then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed abso ⁇ tion of a parenterally administered formulation is accomplished by dissolving or suspending the nitric oxide mimetic in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the nitric oxide mimetic in liposomes or microemulsions which are compatible with body tissues.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations can also be prepared by entrapping the nitric oxide mimetic in liposomes or microemulsions which are compatible with body tissues.
  • Formulations for rectal or vaginal administration are preferably suppositories, sponges or rings, which can be prepared by mixing the nitric oxide mimetics with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity thereby releasing the nitric oxide mimetic.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity thereby releasing the nitric oxide mimetic.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the nitric oxide mimetic is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; humectants such as glycerol; disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents such as paraffin; abso ⁇ tion accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the nitric oxide mimetic only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • Powders and sprays can contain, in addition to the nitric oxide mimetic, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons or alternative non CFC propellants such as DIMEL, also referred to as 1,3,4-A.
  • Dosage forms for topical or transdermal administration of nitric oxide mimetics include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the nitric oxide mimetic is admixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives and/or buffers as may be required.
  • Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • Transdermal patches have the added advantage of providing controlled delivery of the nitric oxide mimetic to the body. Such dosage forms can be made by dissolving or dispensing a nitric oxide mimetic in the proper medium.
  • Abso ⁇ tion enhancers can also be used to increase the flux of the nitric oxide mimetic across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the nitric oxide mimetic in a polymer matrix or gel.
  • the ointments, pastes, creams and gels may contain, in addition to a nitric oxide mimetic, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • a preferred mode of delivery is one which provides a reasonably steady-state delivery of the nitric oxide mimetic, so as to maintain steady-state plasma concentrations. Such delivery avoids any substantial initial spike in plasma concentration of the agent, as it would be desirable to avoid plasma concentrations that produce negative side effects.
  • Transdermal patches and pulsed delivery systems are preferred modes of delivery.
  • the low dose formulations for use in the method of the present invention are preferably formulated according to the same methods as the commercially available higher dose formulations, but with lower amounts sufficient to increase, restore or maintain nitric oxide mimetic activity to cells at a level which inhibits or prevents malignant cell phenotypes and/or enhances the efficacy of an antimalignant therapeutic modality.
  • Methods of formulation are within the skill of pharmaceutical formulation chemists and are fully described in such works as Remington 's Pharmaceutical Science, 18 th Edition, Alfonso R. Gennaro, Ed., Mack Publishing Co., Easton, Pennsylvania, USA, 1990.
  • the methods and formulations of the present invention are particularly useful in inhibiting metastases and development of resistance of tumor cells to antimalignant therapeutic modalities including, but not limited to chemotherapeutic agents, radiation therapy, immunotherapies, and thermal therapies.
  • chemotherapeutic agents useful in combination with low dose NO mimetics include, but are not limited to: anti-angiogenic agents including, but not limited to anti-VEGF agents, alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines.
  • antimetabolites such as folate antagonists, purine analogues, and pyrimidine analogues
  • antibiotics such as anthracyclines, bleomycins, dauxorubicin, mitomycin, dactinomycin, and plicamycin
  • endothelin activating agents enzymes such as L-asparaginase; farnesyl-protein transferase inhibitors; 5 ⁇ reductase inhibitors; inhibitors of 17 ⁇ -hydroxy steroid dehydrogenase type 3; hormonal agents such as glucocorticoids, estrogen or antiestrogens, androgens or antiandrogens, progestins, and luteinizing hormone-releasing hormone antagonist; octreotide acetate; microtubule- disruptor agents, such as ecteinascidins and analogs and derivatives thereof; microtubule- stabilizing agents such as taxanes, for example, TAXOL (paclitaxel),
  • chemotherapeutic agents in these classes useful in the present invention include but are not limited to, actinomycin D, aflacon, bleomycin sulfate, buserelin, busulfan, carmustin, chlorambucil, cladribin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, discodermolides, doxorubicin hydrochloride, estramustine, estramustine phosphate sodium, etoposide, etoposide phosphate, fludarabine, fluorouracil, flutamide, idarubicin, ifosfamide, interferon, interleukins, leuprolide, levamisole, lomustine, mechlorethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin C, paclitaxel, pentastatin, pteridine, quinocarcins, r
  • irinotecan CPT-11; Camptosar
  • isotretinoin isotretinoin (Accutane ; 13-cis-retinoic acid), letrozole (Femara), mechlorethamine (Mustargen), megestrol acetate (Megace), mesna (Mesnex; 2-mercaptoethanesulfonic acid), mitomycin-C (Mutamycin), mitoxantrone (Novantrone; DHAD; DHAQ), nilutamide (Nilandron), oxaliplatin (Eloxatin; DACH- platinum), pemetrexed (Alimta; LY231514), pentostatin (dCF; Nipent; 2 '- deoxycoformycin), procarbine (Matulane; Natulanar; N-Methylhydrazine), raltitrexed (Tomudex; ZD1694), sargramostim (Le
  • Animals suffering from cancer can be administered a low dose of a nitric oxide mimetic to inhibit the metastatic potential of the tumor cells as well as to enhance the efficacy of a co-administered antimalignant therapeutic modality targeted at killing the cancer cells.
  • the nitric oxide mimetic can be administered to animals in combination with other antimalignant therapeutic modalities, following, prior to or during surgical removal or a tumor, and/or following, during, or prior to radiation or thermal therapy. It is believed that this therapy will also enhance the efficacy of anti- NEGF agents targeted at inhibiting angiogenesis of vascular endothelial cells to tumors.
  • low dose nitric oxide mimetic therapy can be administered to an animal prior to, with, or following administration of an anti-NEGF agent such as anti- NEGF antibody.
  • an anti-NEGF agent such as anti- NEGF antibody.
  • the nitric oxide therapy be maintained at least throughout the known active period of the anti-VEGF agent.
  • Low dose nitric oxide mimetic therapy can also be administered as prophylactic therapy to animals at high risk for developing cancer to prevent the development of cells with a malignant cell phenotype.
  • a low dose of the nitric oxide mimetic may be administered daily to the animal throughout its life. Accordingly, administration of long-term sustained release dosing formulations may be preferred in these animals.
  • low dose nitric oxide therapy can be administered to animals suspected of, or known to be, exposed to a factor which lowers cellular nitric oxide mimetic activity so as to induce cells with a malignant cell phenotype.
  • Administration of this low dose nitric oxide therapy is expected to inhibit development of a malignant cell phenotype in these animals.
  • the animal prior to or during a surgical procedure and/or administration of an anesthetic agent in an animal, the animal can also be administered a low dose of a nitric oxide mimetic to prevent and inhibit a malignant cell phenotype.
  • the nitric oxide mimetic be administered for at least the time in which the animal is undergoing the surgical procedure and/or is under the effects of anesthesia.
  • an animal subjected to physical trauma especially a physical trauma associated with blood loss, a decrease in blood volume or hemorrhage can be administered a low dose of a nitric oxide mimetic to prevent and inhibit a malignant cell phenotype.
  • nitric oxide mimetic co- administration of a low dose of a nitric oxide mimetic can also be used to inhibit or prevent a malignant cell phenotype which may occur upon administration of pharmacological agents which alter the circulation, e.g. antihypertensives.
  • the nitric oxide mimetic is preferably administered on a daily basis with the other agents or in a long-term sustained release formulation which extends over the period in which the other agent is administered.
  • Tissue culture medium (RPMI 1640) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY). Hypoxic conditions were generated using airtight chambers from BellCo Biotechnology (Vineland, NJ). GTN was obtained as a solution (TRIDIL, 5 mg ml "1 or 2.22 M) in ethanol, propylene glycol and water (1:1:1.33) from DuPont Pharmaceuticals (Scarborough, ON). Sodium nitroprusside
  • RNA extractions were conducted using a PURESCRIPT RNA isolation kit from Gentra Systems (Minneapolis, MN).
  • the nylon membranes used for the RNA transfers were purchased from Micron Separations, Inc. (Westboro, MA); the uPAR and PAI-1 cDNA probes were cloned in a Bluescript plasmid vector; the [ 32 P]-dCTP and the
  • Reflection NEF film were purchased from Dupont/New England Nuclear (Mississauga, ON); and the oligolabelling kit was obtained from Pharmacia Biotech (Piscataway, NJ).
  • the serum-free EX-CELL 300 culture medium was purchased from JRH Biosciences (Lenexa, KS), the Costar TRANSWELL inserts (6.5 mm diameter polycarbonate, membrane, 8 ⁇ m pore) were purchased from Coming Costar
  • anti-uPAR antibody monoclonal antibody [MoAb] 3937
  • ASDL enhanced chemiluminescence
  • the gelatin was purchased from BDH (Toronto, ON)
  • the casein was bought from Sigma Chemical Co. (St. Louis, MO)
  • the plasminogen was from American Diagnostica (Greenwich, CT).
  • HTR-8/SNneo invasive trophoblast cell line and the MDA-MB-231 metastatic breast carcinoma cell line were used in these experiments. Both the HTR- 8/SNneo and the MDA-MB-231 cells were cultured in RPMI-1640 medium supplemented with 5% FBS.
  • the HTR-8/SNneo cell line was obtained from explant cultures of human first trimester placenta and immortalized by transfection with a cD ⁇ A construct encoding the
  • SNneo large T antigen These cells have been previously characterized and have been maintained in culture for over 130 passages in RPMI 1640 medium supplemented with 5% FBS. They exhibit a high proliferation index and share various phenotypic similarities with the non-transfected parent HTR-8 cells such as in vitro invasive ability and lack of tumorigenicity in nude mice .
  • the MDA-MB-231 cell line was initially isolated in 1973 from the pleural effusion of a 51- year-old breast cancer patient (Callieau et al J. Nat. Cancer Inst. 1974 53:661-674).
  • GTN Glyceryl Trinitrate
  • SNP Sodium Nitroprusside
  • the cells were treated with varying concentrations of GTN and SNP.
  • the stock solution of GTN was first diluted in phosphate-buffered saline (PBS) to a concentration of 10 "4 M. Following filtration, the GTN solution was diluted in the culture medium to concentrations ranging from 10 "4 M to 10 " " M.
  • the SNP originally in crystal form was dissolved in distilled water and diluted to a concentration of 10 "5 M. Following filtration, the SNP was diluted in the culture medium to concentrations ranging from 10 "6 M, to 10 "12 M.
  • the cells were first cultured with 20% O 2 or 1% O 2 in the presence of varying concentrations of SNP or GTN. Following incubations, the cells were lysed using a buffer containing 40 mM HEPES pH 7.2, 100 mM NaCI, 20% glycerol, 0.1 mM EDTA pH 8.0, 0.2% Triton X-l 00, 1 mM DTT, and 2 mM PMSF. The lysates were then subjected to homogenization, DNA shearing (10 times with a 25 V 8 -gauge needle), boiling (5 minutes) and centrifugation (15 minutes, 14000g). The supernatant was collected and stored at -80°C until use.
  • the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and the resolved proteins were transferred to an Immobilon-P membrane using a wet transfer apparatus (Bio-Rad Laboratories, Mississauga, ON).
  • the membranes were blocked overnight at 4°C in a solution containing 1% PBS, and 0.01 % Tween20 (PBS-T) as well as 5% casein.
  • the blots were subsequently incubated for 1.5 hours with the monoclonal anti-uPAR antibody [MoAb 3937], followed by six 5 minute washes with PBS-T.
  • the membranes were then incubated with a horseradish peroxidase labeled goat anti-mouse IgG secondary antibody for 1.5 hours. Following six additional 5 minute washes with PBS-T, the antigen was detected by enhanced chemiluminescence and the blots were exposed onto Dupont
  • the gel contained 0.1%) w/v gelatin, and for the analysis of plasminogen activator secretion, the gel was supplemented with 0.1 % w/v casein as well as plasminogen (50 ⁇ g/mL).
  • the serum-free conditioned medium was then combined with a nonreducing sample loading buffer (0.5 M Tris, 10% SDS, 1% Bromophenol Blue in 2 mL glycerol) in a ratio of 4:1 and was not boiled.
  • a nonreducing sample loading buffer 0.5 M Tris, 10% SDS, 1% Bromophenol Blue in 2 mL glycerol
  • gelatin-containing gels these areas corresponded to metalloproteinase (gelatinase) activity and in the casein gels, these bands corresponded to plasminogen activator activity.
  • the gels were preserved using a preserving solution (10% glacial acetic acid/10% glycerol/80% distilled water) for 1 hour and were dried on cellophane for 1 hour at 60°C.
  • Matrigel invasion chambers (modified Boyden chambers) were used to assess the invasive ability of the cells under hypoxic and standard conditions in the presence or absence of various concentrations of GTN or SNP.
  • the chambers consist of cell culture inserts, 6.5 mm in diameter and with a 8 ⁇ m pore size membrane. Each membrane was coated with 100 ⁇ L of a 1 mg/mL solution of Matrigel diluted in cold serum-free culture medium (EX-CELL 300), and allowed to dry in a laminar flow cabinet for approximately 12 hours. The Matrigel was then rehydrated by incubating it with 100 ⁇ L of serum-free medium for approximately 1 hour. After rehydration, cell suspensions containing 5.0 x
  • the membrane was removed from the insert housing with a small scalpel blade, mounted onto a microscope slide and coverslipped. Invading cells were then viewed under the microscope at 40X magnification and counted. The invasion index for each treatment was calculated by dividing the number of invading cells by the number of cells which invaded under standard conditions. This value was then multiplied by 100 to obtain a percentage. The standard was given a value of 100%> and the treatment values were converted to a percentage of the standard. The results were tested for statistical significance using either the Tukey test for pair-wise multiple comparison procedures or the Student-Newman- Keuls method for pair- wise multiple comparison procedures. See Figure 1.
  • mice were injected i.v. (tail vein) with a bolus of 5 x 10 4 - 10 5 B 16F 10 metastatic melanoma cells.
  • mice were randomly divided into groups of 15 and mice in each group were placed in plexiglass chambers (approximately 3 L) which were continuously flushed with gas mixtures of 20% O 2 :balance N 2 and 10% O 2 :balance N 2 , respectively. Gas flows were adjusted to a level which did not allow CO 2 build-up within the chambers.
  • mice After a 24 hour exposure to an atmosphere of either 20% O 2 or 10% O 2 , mice were removed from the chambers and placed in regular cages kept at room air. Thirteen days later, mice were sacrificed by cervical dislocation, and lungs were removed and fixed in Bouin's fixative (Sigma). Metastatic nodules (many of which appeared black due to the presence of melanin) on the surface of the lungs were counted visually under a dissecting microscope. Data were expressed as the number of lung nodules per 10 4 cells injected and were analyzed using statistical tests for non-parametric values.
  • MDA-MB-231 breast cancer cells The resistance of MDA-MB-231 breast cancer cells to doxorubicin was determined following culture in 20% or 1% oxygen by counting the number of colonies formed. MDA-MB-231 cells were incubated in 1% O 2 or 20% O 2 for 24 hours. Following incubation, the cells were exposed to diluent (control), 25 ⁇ M doxorubicin, 25 ⁇ M doxorubicin plus 10 "6 M GTN or 25 ⁇ M doxorubicin plus 10 "10 M GTN for 1 hour.
  • the cells were washed and then plated onto 35 mm plates at different dilutions. The cells were incubated for an additional 1-2 weeks in order to allow cell colonies to grow. At the end of the incubation period, the cells were fixed with Camoy's fixative, stained with Crystal violet, rinsed and allowed to air dry. Colonies were counted visually. The surviving cells under each condition was determined by counting the number of colonies and was expressed as a fraction of the number of colonies that survived without doxorubicin exposure.
  • GTN Glyceryl Trinitrate
  • DETA-NONOate Diethylenetriamine/nitric oxide adduct
  • the HTR-8/SVneo line was established from explant cultures of human first trimester placenta and was immortalized by transfection with the large T antigen of simian virus 40. These cells exhibit similar in vitro invasive abilities to their parent HTR-8 cells, and are not tumourigenic in nude mice. They have been maintained in culture for over 130 passages, and require at least 5% se m in order to sustain growth in vitro (see, Graham, Hawley, et al. 1993 ID: 1080).
  • the MDA-MB-231 is a metastatic breast cancer cell line that was initially isolated in 1973 from a 51-year-old patient ⁇ Cailleau, Young, et al 1974 ID: 982 ⁇ .
  • a metastatic murine melanoma (B 16 F 10) (a kind gift of Dr. Ann Chambers, London Regional Cancer Centre, London, ON) was utilized in the experimental metastasis experiments.
  • All of the cell cultures were maintained in monolayer culture in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 5% fetal bovine semm (FBS; Gibco BRL) in a standard Sanyo CO 2 incubator (5 % CO 2 in air, 37°C; Esbe Scientific, Markham, ON).
  • RNA was isolated using a GENTRA PURESCRIPT ® RNA Isolation Kit (Gentra Systems, Minneapolis, MN). The isolated RNA was subsequently separated by electrophoresis, transferred to a charged nylon membrane (Micron Separations Inc., Westboro, MA) and fixed with ultraviolet radiation using a UV Crosslinker (NAME). The membranes were prehybridized at 68°C for approximately 1 hour in a prewanned ultrasensitive hybridization buffer, ULTRAhyb TM (Ambion, Austin, TX). They were then hybridized overnight at 68°C with a DIG-labeled uPAR probe.
  • GENTRA PURESCRIPT ® RNA Isolation Kit Genetra Systems, Minneapolis, MN.
  • NAME UV Crosslinker
  • a uPAR cDNA cloned in a Bluescript plasmid vector was linearized.
  • the linear cDNA subsequently underwent in vitro transcription with a DIG-labelled RNA labeling mix and T3 RNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). Following hybridization, the membranes were washed twice in 2X SSC, 0.1 % SDS (5 minutes at 68°C) and then twice in 0.1X SSC, 0.1 % SDS (15 minutes at 68°C). Bands were detected using a DIG luminescent detection kit (Roche Molecular Biochemicals, Indianapolis, IN) and the membrane was exposed to Dupont Reflection NEF film (Dupont / New England Nuclear, Mississauga, ON).
  • the cells were lysed using a buffer containing 40 mM HEPES pH 7.2, 100 mM NaCI, 20% glycerol, 0.1 mM EDTA pH 8.0, 0.2% Triton X- 100, 1 mM dithiothreitol (DTT), and 2 mM phenylmethyl sulfonyl fluoride (PMSF).
  • the lysates were then subjected to homogenization, DNA shearing (10 times with a 25 5 / 8 - gauge needle), boiling (5 minutes) and centrifugation (15 minutes at 14,000 x g). The supernatant was collected and stored at -80°C until use.
  • the samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and the resolved proteins were transferred to an Immobilon-P membrane (Millipore Co ⁇ ., Bedford, MA) using a wet transfer apparatus (BioRad Laboratories, Mississauga, ON).
  • the membranes were blocked overnight at 4°C in a solution containing 1% PBS, and 0.01% Tween 20 (PBS- T) as well as 5% dry milk powder.
  • the blots were subsequently incubated for 1.5 hours with the monoclonal anti-uPAR (MoAb 3937; American Diagnostica Inc., Greenwich,
  • Matrigel invasion assays were used to assess the effect of 8-Br-cGMP on the ability of cells to penetrate through an extracellular matrix.
  • the chambers consisted of 6.5-mm inserts with 8- ⁇ m pore membranes (Coming Costar Co ⁇ ., Cambridge, MA). Each membrane was coated with 100 ⁇ L of a 2.6 mg/mL solution of Matrigel (Collaborative Biochemical Products, Bedford, MA) diluted in cold serum-free medium
  • a murine metastasis model was used to assess the effect of 8-Br-cGMP on the metastatic potential of B16-F10 melanoma cells.
  • the cells were incubated for 12 hrs in 1% or 20% oxygen and in the presence or absence of 8-Br-cGMP (1 ⁇ M).
  • 8-Br-cGMP 1 ⁇ M
  • 5 x 10 4 cells were injected intravenously (tail vein injections) into 5-7 week old C57B1/6 female mice.
  • Cells were also plated in tissue culture plates to determine the effect of culture conditions on in vitro colony forming ability. Fourteen days after intravenous inoculation, mice were sacrificed and their lungs were removed and fixed in Bouin's fixative.
  • cGMP levels were measured. Briefly, cells were extracted over ice in 1 mL of 6 % trichloroacetic acid (BDH Laboratory Supplies, Poole, England). The homogenate was then centrifuged at 13,000 g for 10 min. The supernatant fraction was removed and extracted five times with 2mL of water-saturated diethyl ether (BDH). The cGMP contained in this fraction was subsequently acetylated and quantitated using an enzyme-linked immuno-sorbent assay (ELISA) kit (STI - Signal Transduction Products, San Clemente, CA).
  • ELISA enzyme-linked immuno-sorbent assay
  • the selective sGC inhibitor ODQ (0.5 ⁇ M) was used to determine the role of sGC in the NO mimetic prevention of uPAR upregulation. It was found that the ability of GTN (10 "6 M) to abrogate the hypoxic induction of uPAR was substantially prevented by
  • MDA-MB-231 cells were cultured in 0.5%> oxygen for 24 hours to determine the effect on uPAR protein levels. It was found that exposure to 0.5% oxygen resulted in up to 3.5-fold increases (P ⁇ 0.0001) in uPAR protein levels ( Figures 6A and 9B) and up to 5-fold increases (P ⁇ 0.0002) in uPAR mRNA levels ( Figures 6C, 8A, and 9A).
  • MB-231 cells were incubated for 24 hours with the selective sGC blocker (ODQ, 0.5 ⁇ M). Compared with incubation of cells under control conditions (20% O 2 alone), the treatment described above resulted in a 2.7-fold increase (p ⁇ 0.05) in the levels of uPAR mRNA ( Figure 8 A). While the presence of GTN (1 ⁇ M) prevented the hypoxic upregulation of uPAR mRNA expression, GTN was unable to block the effect of hypoxia when ODQ was also present in the medium ( Figure 8 A).
  • Soluble GC is a heme-containing enzyme that requires ferrous iron for its biosynthesis and activity.
  • Example 3 The role of nitric oxide in hypoxia-induced chemoresistance in prostate cancer cells
  • hypoxia low tumor oxygenation conelates with increased metastasis and resistance to radiotherapy and chemotherapy.
  • Hypoxia has been shown to be associated with human prostate cancer, and prostate cancers are often highly resistant to chemotherapy.
  • hypoxia is a major contributor to reduced chemosensitivity in prostate cancer cells and that hypoxia-induced chemoresistance in prostate cancer cells can be inhibited by low concentrations of nitric oxide (NO) mimetic agents
  • human PC-3 and mouse TRAMP-C2 prostatie adenocarcinoma cells were incubated in 20% O 2 or 0.5% O 2 for 12 hours in the presence or absence of glyceryl trinitrate (GTN, 0.1 nM).
  • GTN glyceryl trinitrate
  • Cervial cancer is most common in developing countries.
  • the cervix is the lower third of the utems. Cancer of the cervix may originate on the vaginal surface or in the canal. Each year, an estimated 500,000 new cases are diagnosed worldwide with some 250,000 of those women destined to die of their disease. Ample evidence exists to show that both incidence and mortality can be reduced by the use of cervical screening program (Cain JM, et al, Science, 288:1753-1754, 2000). Cunently, human papillomaviras (HPV), especially HPVs 16 & 18, has been implicated as the major causal agent in this disease.
  • HPV human papillomaviras
  • Symptoms of cervical cancer include vaginal bleeding, post-coital spotting, vaginal discharge, and in advanced cases, pelvic or low back pain with sciatic nerve toot type of pain radiating down to the lower back of the lower extremities.
  • PAP smears which analyses the cytological abnormalities of the cervical epithelial cells.
  • a National Institute of Health consensus panel was formed and had a uniformed terminology for reporting cervical cytology.
  • Low grade squamous intraepithelial lesions (LSIL) encompassing HPV changes are considered Grade I CIN.
  • High-grade lesions encompassing Grade II & III CIN present high risk to progress cervical cancer.
  • the next step could be colposcopy and directed biopsy.
  • diagnosis is usually confirmed by a directed cervical punch biopsy (Benedet JL et al, International Journal of Gynecology & Obstetrics, 70 (2000), 209-262).
  • Example 8 Prolong the Effective Treatment Period and Enhance the Overall Effectiveness of Treating Hormone Refractory/insensitive Breast Cancer Using Fourth-Line Treatment Option by Oral PDE V Inhibitor
  • breast cancer is the most common cancer and the second leading cancer death in women. In the United States, there are approximately 100,000 women alive with metastatic breast cancer. Once metastases have developed, breast cancer is rarely, if ever, a "curable" condition.
  • the treatment objectives are to optimize treatment response, improve survival, and balance these goals with maintaining highest possible quality of life. Some treatments are palliative in nature. With the exception of Herceptin (specific for the HER2 oncoprotein), cytotoxic chemotherapeutic agents remains the standard for advanced hormone insensitive breast cancer.
  • Enhancing the quality of life, reduction of pain and discomfort during the survival period is also considered as support of Viagra effectiveness in treating breast cancer patients using fourth-line treatment approach.
  • Viagra could also be used with glucocorticoids such as prednisone to provide additional palliative relief of cancer symptoms.
  • Prostate cancer is an important public health concern, representing the most common visceral cancer and the second leading cause of cancer deaths of North American males. In 1999, the American Cancer Society estimated that approximately 179,300 new cases of prostate cancer will be diagnosed in the United States and about
  • Radical prostatectomy is considered a gold therapy treatment of localized prostate cancer and removal of all prostatie tissue should result in undetectable semm PSA within a month if all disease has been eradicated (Landis et al. CA Cancer J Clin 1999 49:8-31); therefore, longitudinal measurement of PSA is cunently the most sensitive method for detecting cancer persistence, relapse and progression following radical prostatectomy
  • the median time to PSA evidence of treatment failure ranges from 19 to 24 months and the median interval from biochemical recurrence to clinical evidence of disease is an additional 19 months (range: 7 to 71 months).
  • Men diagnosed with prostate cancer can be treated with a low-dose NO mimetic such as nitroglycerin or isosorbide dinitrate, preferably orally, sublingually, topically, or parenterally, alone or in combination with the standard therapies (e.g., anti-androgen therapy, radical prostatectomy).
  • exemplary low-dose NO mimetic therapies are set forth herein in Table 1. Regression or stabilization of serial PSA values is considered a treatment success.
  • a low-dose NO mimetic such as nitroglycerin or isosorbide dinitrate treatment
  • Subjects in the high-risk group e.g., with short PSA doubling times or family history can stay on a low-dose NO mimetic (e.g., nitroglycerin or isosorbide dinitrate) treatment indefinitely to prevent development, progression or recurrence of the disease.
  • a low-dose NO mimetic e.g., nitroglycerin or isosorbide dinitrate
  • Females diagnosed with breast cancer can be treated with a low-dose NO mimetic such as nitroglycerin or isosorbide dinitrate, preferably orally, sublingually, topically, or parenterally, alone or in combination with the standard chemotherapies (e.g. taxol).
  • a low-dose NO mimetic such as nitroglycerin or isosorbide dinitrate
  • exemplary low-dose NO mimetic therapies are set forth herein in Table 1. Regression or stabilization of a primary tumor is considered a treatment success.
  • Subjects in the high- risk group e.g. family history
  • a low-dose NO mimetic e.g. nitroglycerin or isosorbide dinitrate
  • PSA levels a biochemical marker of prostate cancer recunence, were determined following GTN administration in patients with prostate cancer wherein the prostate is still intact.
  • Prostate cancer is now the most commonly diagnosed cancer and the second most common cause of cancer death in men.
  • Prostate cancer is mostly adenocarcinoma that arise from high-grade prostate intraepithelial neoplasia (PIN) which is present in 4-16.5%o of needle biopsies and is strongly predictive of co-existing carcinoma, thus wananting a repeat biopsy.
  • PIN prostate intraepithelial neoplasia
  • Early prostate cancer patients can be effectively treated with available androgen suppression therapy.
  • hormone refractory advanced prostate cancer remains a clinical challenge.
  • HRPC hormone-refractory/insensitive prostate cancer
  • patients who failed one non-estramustine, taxane-containing regimen and entering an estramustine treatment regimen e.g. 14mg/kg/day PO in 3-4 individual doses
  • an estramustine treatment regimen e.g. 14mg/kg/day PO in 3-4 individual doses
  • the nitroglycerin ointment will be applied 4-5 times daily to provide sustained plasma level of nitric oxide.
  • the PSA levels While under the nitroglycerin and estramustine treatment, the PSA levels will be measured monthly.
  • the effect of nitroglycerin treatment will be measured against historical database of estramustine users.
  • a statistic of > 40% patients with > 50%> decrease in PSA levels or velocity will be considered a nitroglycerin treatment success.
  • the overall increase in the expected median survival time of 5 months of estramustine mono-therapy, the reduction of pain score and the use of pain medication, or improvement in patients' ability to maintain cognitive function and to copy with the advanced disease will also considered as clinically meaningful to add nitroglycerin treatment to the standard of care of these patients.
  • Patients with localized early stage non-metastatic breast cancer may elected to undergo a lumpectomy procedure to remove the malignant tissue.
  • patients Prior to the surgery, patients may start with first line anti-malignant therapy to reduce the tumor volume.
  • patients could start taking nitric oxide mimetics such as Viagra (25mg or 50 mg) daily continuously during the first line therapy, right after the surgery and chronically post-surgical procedure to prevent relapse of the breast cancer or other form of cancer. Prolong remission period against the history database and/or better quality of life will be considered a treatment success.

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Abstract

L'invention concerne des procédés et des formulations permettant d'inhiber, de traiter et de prévenir un phénotype cellulaire malin, une cellule, une tumeur et/ou une maladie. L'administration de substances mimétiques d'oxyde nitrique, en faibles doses, suffit pour accroître, restaurer ou maintenir la signalisation induite par l'oxyde nitrique dans des cellules, de manière que ces phénotypes cellulaires malins, ces cellules, ces tumeurs et/ou ces maladies soient inhibés ou prévenus. Ces procédés et formulations sont particulièrement utiles pour le traitement et la prévention du cancer chez des animaux.
PCT/CA2003/000313 2002-03-06 2003-03-06 Formulations et procedes d'utilisation de substances mimetiques de l'oxyde nitrique dans le traitement du cancer WO2003074082A1 (fr)

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JP2003572598A JP2005527510A (ja) 2002-03-06 2003-03-06 癌の治療において一酸化窒素模倣体を使用するための製剤および方法
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WO2006018088A1 (fr) * 2004-08-19 2006-02-23 Switch Biotech Ag Utilisation d'un inhibiteur pde5 aux fins de traitement et de prevention de troubles hypopigmentaires
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US7329495B2 (en) 2004-06-09 2008-02-12 Board Of Regents, The University Of Texas System Mutations in KIT confer imatinib resistance in gastrointestinal stromal tumors
JP2008509944A (ja) * 2004-08-19 2008-04-03 スイッチ ビオテッヒ アーゲー 低色素性障害を治療・予防するためのｐｄｅ５阻害剤、異性体、塩の使用
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US11229686B2 (en) 2015-09-28 2022-01-25 Alexion Pharmaceuticals, Inc. Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia
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US11352612B2 (en) 2015-08-17 2022-06-07 Alexion Pharmaceuticals, Inc. Manufacturing of alkaline phosphatases
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US7329495B2 (en) 2004-06-09 2008-02-12 Board Of Regents, The University Of Texas System Mutations in KIT confer imatinib resistance in gastrointestinal stromal tumors
JP4729503B2 (ja) * 2004-06-10 2011-07-20 日本化薬株式会社 抗癌剤の奏効率増強剤
JP2008501630A (ja) * 2004-06-10 2008-01-24 日本化薬株式会社 抗癌剤の効果増強剤
JP2011144190A (ja) * 2004-06-10 2011-07-28 Nippon Kayaku Co Ltd 抗癌剤の効果増強剤
US8410175B2 (en) 2004-06-10 2013-04-02 Nippon Kayaku Kabushiki Kaisha Anticancer effect enhancer
WO2005120493A1 (fr) * 2004-06-10 2005-12-22 Tohoku University Promoteur de l'effet anticancereux
JP2008509944A (ja) * 2004-08-19 2008-04-03 スイッチ ビオテッヒ アーゲー 低色素性障害を治療・予防するためのｐｄｅ５阻害剤、異性体、塩の使用
EP1759700B1 (fr) * 2004-08-19 2009-08-05 Switch Biotech, LLC Utilisation d'inhibiteurs de PDE5 pour traiter et prévenir les maladies liées à la hypopigmentation
WO2006018088A1 (fr) * 2004-08-19 2006-02-23 Switch Biotech Ag Utilisation d'un inhibiteur pde5 aux fins de traitement et de prevention de troubles hypopigmentaires
JP4904268B2 (ja) * 2004-08-19 2012-03-28 スイッチ バイオテック エルエルシー 低色素性障害を治療・予防するためのｐｄｅ５阻害剤、異性体、塩の使用
US8268785B2 (en) * 2004-08-19 2012-09-18 Switch Biotech Ag Use of a Pde 5 inhibitor for treating and preventing hypopigmentary disorders
US8329656B2 (en) 2004-08-19 2012-12-11 Switch Biotech Ag Use of a PDE5 inhibitor for treating and preventing hypopigmentary disorders
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US11224638B2 (en) 2014-12-05 2022-01-18 Alexion Pharmaceuticals, Inc. Treating seizure with recombinant alkaline phosphatase
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US11229686B2 (en) 2015-09-28 2022-01-25 Alexion Pharmaceuticals, Inc. Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia
US11400140B2 (en) 2015-10-30 2022-08-02 Alexion Pharmaceuticals, Inc. Methods for treating craniosynostosis in a patient
US11065306B2 (en) 2016-03-08 2021-07-20 Alexion Pharmaceuticals, Inc. Methods for treating hypophosphatasia in children
US11186832B2 (en) 2016-04-01 2021-11-30 Alexion Pharmaceuticals, Inc. Treating muscle weakness with alkaline phosphatases
US10898549B2 (en) 2016-04-01 2021-01-26 Alexion Pharmaceuticals, Inc. Methods for treating hypophosphatasia in adolescents and adults
US10988744B2 (en) 2016-06-06 2021-04-27 Alexion Pharmaceuticals, Inc. Method of producing alkaline phosphatase
US11116821B2 (en) 2016-08-18 2021-09-14 Alexion Pharmaceuticals, Inc. Methods for treating tracheobronchomalacia
US11224637B2 (en) 2017-03-31 2022-01-18 Alexion Pharmaceuticals, Inc. Methods for treating hypophosphatasia (HPP) in adults and adolescents
US11913039B2 (en) 2018-03-30 2024-02-27 Alexion Pharmaceuticals, Inc. Method for producing recombinant alkaline phosphatase
CN114869894A (zh) * 2022-05-10 2022-08-09 福州大学 一种小分子化合物在制备作为尿激酶受体抑制剂药物中的应用

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