US 2005O142217A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0142217 A1 Adams et al. (43) Pub. Date: Jun. 30, 2005

(54) FORMULATIONS AND METHODS OF USING Related U.S. Application Data NITRIC OXDE MIMETCS AGAINST A MALIGNANT CELL PHENOTYPE (63) Continuation-in-part of application No. 09/842,547, filed on Apr. 26, 2001. (76) Inventors: Michael A. Adams, Kingston (CA); Charles H. Graham, Kingston (CA); (60) Provisional application No. 60/277.469, filed on Mar. Jeremy P.W. Heaton, Ganonoacque 21, 2001. Provisional application No. 60/199,757, (CA); Lynne-Marie Postovit, Kingston filed on Apr. 26, 2000. (CA) Publication Classification Correspondence Address: TOWNSEND AND TOWNSEND AND CREW, (51) Int. Cl...... A61K 33/00 LLP (52) U.S. Cl...... 424/718 TWO EMBARCADERO CENTER EIGHTH FLOOR SAN FRANCISCO, CA 94111-3834 (US) (57) ABSTRACT (21) Appl. No.: 10/042,039 Methods and formulations for inhibiting and preventing a malignant cell phenotype by administering to cells a low (22) Filed: Oct. 25, 2001 dose of a nitric oxide mimetic are provided. Patent Application Publication Jun. 30, 2005 Sheet 1 of 6 US 2005/0142217 A1

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FORMULATIONS AND METHODS OF USING tumor growth and progression. For example, nitric oxide NITRIC OXDE MIMETCS AGAINST A generation by inducible nitric oxide synthase (iNOS) has MALIGNANT CELL PHENOTYPE been implicated in the development of prostate (Klotz et al. Cancer; National Library of Medicine, MDX INTRODUCTION Health Digest 199882(10): 1897-903), as well as in colonic adenocarcinomas and mammary adenocarcinomas (Lala, P. 0001. This application is a continuation in part of U.S. K. and Orucevic, A., Cancer and Metastasis Reviews 1998 application Ser. No. 09/842,547, filed Apr. 26, 2001, which 17:91-106). In addition, nitric oxide has been suggested to claims the benefit of priority from U.S. Provisional Appli play an important role in the metabolism and behavior of cation Ser. No. 60/277.469 filed Mar. 21, 2001, and U.S. lung , and in particular adenocarcinomas (Fujimoto Provisional Application Ser. No. 60/199,757 filed Apr. 26, et al. Jpn. J. Cancer Res 1997 88:1190-1198). In fact, it has 2000, which are herein incorporated by reference. been Suggested that tumor cells producing or exposed to what these researchers refer to as low levels of nitric oxide, FIELD OF THE INVENTION or tumor cells capable of resisting nitric oxide-mediated 0002 The present invention relates to methods and for injury undergo a clonal Selection because of their Survival mulations for inhibiting and preventing a malignant cell advantage (Lala, P. K. and Orucevic, A. Cancer and Metasta phenotype. We have now found that the mechanism by sis Review 1998 17.91-106). These authors suggest that which hypoxia and hyponitroXia have impact upon cellular these tumor cells utilize certain nitric oxide-mediated phenotype is not necessarily mediated Solely by the lack of mechanisms for promotion of growth, invasion and metasta oxygen but rather also from a deficiency in nitric oxide sis and propose that nitric oxide-blocking drugs may be mimetic activity. Accordingly, as demonstrated herein useful in treating certain human cancers. There is also administration of low doses of nitric oxide mimetics is evidence indicating that tumor-derived nitric oxide promotes Sufficient to increase, restore or maintain nitric oxide tumor angiogenesis as well as invasiveness of certain tumors mimetic activity of cells So that malignant cell phenotypes in animals, including humans (Lala, P. K. Cancer and are inhibited or prevented. Thus, provided herein are for Metastasis Reviews 1998 17:1-6). mulations and methods of using these formulations to 0006. However, nitric oxide has been disclosed to reverse deliver low doses of nitric oxide mimetics to cells at production of vasoconstrictors induced by hypoxia (Faller, concentrations which inhibit a malignant cell phenotype D. G. Clinical and Experimental Pharmacology and Physi and/or prevent development of a malignant cell phenotype ology 1999 26:74-84). In addition, the nitric oxide donors but which reduce or avoid development of unwanted effects Sodium nitroprusSide, S-nitroSO-L-glutathione and 3-mor of the NO mimetics. These methods and formulations are pholinoSydnonimine in the micromolar range (IC=7.8, 211 particularly useful in treating and preventing cancer in and 490 uM, respectively) have been demonstrated to Sup animals. preSS the adaptive cellular response controlled by the tran Scription factor hypoxia-inducible factor-1 in hypoxically BACKGROUND OF THE INVENTION cultured Hep3B cells, a human hepatoma cell line (Sogawa 0.003 Hypoxia or oxygen tension below normal physi et al. Proc. Natl. Acad. Sci. USA 1998 95:7368-7373). The ologic value in cells results in physiologic as well as nitric oxide donor sodium nitroprusside (SNP; 150 uM) has pathologic alterations in the cells, which alterations have also been demonstrated to decrease hypoxia-induced expres been associated with differential gene expression. For Sion of vascular endothelial growth factor, an endothelial example, hypoxia affects endothelial cellular physiology in cell mitogen required for normal vascular development and Vivo and in vitro in various ways including modulating the pathological angiogenic diseaseS Such as cancer and iris and transcriptionally-regulated expression of vasoactive Sub retinal neovascularization (Ghiso et al. Investigative Oph stances and matrix proteins involved in modulating vascular thalmology & Visual Science 1999 40(6): 1033-1039). In tone or remodeling the vasculature and Surrounding tissue these experiments, 150 uMSNP was demonstrated to com (Faller, D. V. Clin. Exp. Pharmacol. and Physiol. 1999 pletely suppress hypoxia-induced VEGF mRNA levels for at 26:74-84). Hypoxia in solid tumors has been shown to least 24 hours in immortalized human retinal epithelial cells. protect cancer cells from being killed by X-irradiation and 0007 High levels of nitric oxide, when induced in certain leads to resistance to certain cancer drugs. Hypoxia also cells, can cause cytostasis and . For example, Xie appears to accelerate malignant progression and increase et al. have demonstrated exposure to high levels of nitric metastasis (Brown, J. M. Cancer Res. 1999 59:5863-5870). oxide (producing approximately 75 uM nitrite; see FIG. 5A of Xie et al.) to be an exploitable phenomenon to promote 0004 Nitric oxide has been implicated in various bio death (see FIGS. 6A and 6B of Xie et al.) in murine K-1735 logical processes. For example, nitric oxide is a biological melanoma cells (J. Exp. Med. 1995 181:1333-1343). In messenger molecule responsible for endothelium derived addition, WO 93/20806 discloses a method of inducing cell vascular relaxation and neurotransmission. Nitric oxide, at cytostasis or cytotoxicity by exposing cells to a compound what these researchers refer to as high levels, is also known Such as Spermine-bis(nitric oxide) adduct monohydrate at as a mediator for anti-tumor and anti-bacterial actions of 500 uM which is capable of releasing nitric oxide in an macrophages. Nitric oxide has also been demonstrated to aqueous Solution. The compounds are taught to be useful in play a modulatory role on cytokine-induced expression of the treatment of tumor cells as well as in antiparasitic, matrix metalloproteinase-9 and tissue inhibitors of metallo antifungal and antibacterial treatments. Use of a mega proteinases (Eberhardt et al. Kidney International 2000 dosing regimen is Suggested, wherein a large dose of the 57:59-69). nitric oxide releasing compound is administered, time is 0005. A large body of clinical and experimental data allowed for the active compound to act, and then a Suitable indicates that nitric oxide also plays a role in promoting Solid reagent Such as a nitric oxide Scavenger is administered to US 2005/0142217 A1 Jun. 30, 2005

the individual to render the active compound inactive and to malignant cell phenotype to antimalignant therapeutic Stop non-specific damage. It is taught at page 14, line 25-30 modalities as well as increase the efficacy of antimalignant of WO 93/20806 that 3-(n-propyl amino)propylamine bis therapeutic modalities. (nitric oxide) adduct, diethylamine-bis(nitric oxide) adduct Sodium salt, isopropylamine-bis(nitric oxide) adduct Sodium 0013 The methods and formulations of the present Salt, Sodium trioxodinitrate (II) monohydrate, and N-nitroSo invention are also very useful in preventing a malignant cell hydroxylamine-N-sulfonate did not significantly affect cell phenotype which can develop in cells upon exposure of cells to conditions and/or therapeutic agents which lead to a viability at concentrations up to 500 uM. deficiency in nitric oxide mimetic activity in the cells. 0008 U.S. Pat. No. 5,840,759, U.S. Pat. No. 5,837,736, 0014. The methods and formulations of the present and U.S. Pat. No. 5,814,667, disclose methods for using invention are also useful in inhibiting development of a mg/kg quantities of nitric oxide releasing compounds to more aggressive malignant cell phenotype in cancer cells Sensitize hypoxic cells in a tumor to radiation. These patents which can occur upon exposure to factors which induce Such also disclose methods of using the same nitric oxide-releas development. ing compounds at mg/kg levels to protect noncancerous cells or tissue from radiation, to Sensitize cancerous cells to 0015. In addition, these methods and formulations are chemotherapeutic agents, and to protect noncancerous cells useful in diagnosing and monitoring a malignant cell phe or tissue from chemotherapeutic agents. Compounds used in notype in an animal via detection of levels of one or more these methods Spontaneously release nitric oxide under markers indicative of a malignant cell phenotype following physiologic conditions without requiring oxygen. These administration of a low dose of a nitric oxide mimetic. No patents teach administration of the nitric oxide-releasing change, a decrease or deceleration in the increase of the level compound from about 15 to about 60 minutes prior to of one or more of these markers in an animal following therapy. Typical doses of the nitric oxide releasing com administration of a low dose nitric oxide mimetic as com pound administered are Suggested to be from about 0.1 to pared to the level of the marker in the animal prior to about 100 mg of one or more nitric oxide releasing com administration of the low dose nitric oxide mimetic is pounds per kg of body weight. Concentrations of the nitric indicative of a malignant cell phenotype in the animal. oxide releasing compounds DEA/NO and PAPA/NO dem onstrated to increase the sensitivity of MCF7 0016. Accordingly, the methods and formulations of the cells and V79 fibroblasts to melphalan, thiotepa, mitomycin present invention provide new therapeutic and diagnostic C, SR4233 and cisplatin in vitro were in the millimolar approaches for the treatment and prevention of cancer in range while 70 mg/kg of DEA/NO was demonstrated to animals. increase the Survival of mice administered the chemothera peutic agent Melphalan in the in vivo KHT tumor model. BRIEF DESCRIPTION OF THE DRAWINGS 0009 U.S. Pat. No. 5,700,830 and WO 96/15781 disclose 0017 FIG. 1 is a histogram showing the effect of GTN methods for inhibiting adherence between cancerous cells and SNP on the in vitro invasion by MDA-MB-231 invasive and noncancerous cells in an animal by administering to the breast cancer cells in hypoxic (1% O2) conditions as com animal a nitric oxide-releasing compound containing a nitric pared to normal (20% O.) conditions. Cells were coated oxide-releasing NO functional group. Recent studies, onto MATRIGEL-coated membranes and incubated under however, indicate that cancer cell adhesion to and Spreading hypoxic or normal conditions, alone or in the presence of along the vessel wall leading to extravasation is not an nitric oxide mimetics. The invasion index (% of control) which is taken to be a measure of invasive potential of the obligatory event in metastasis (Morris et al. Exp. Cell. Res. cells for each treatment was determined by Staining the cells 1995 219:571-578). which invaded through the membrane and counting them. 0010 WO 98/58633 discloses a microdose nitric oxide The first bar depicts the invasion index of cells cultured therapy for alleviating vascular conditions associated with a under normal conditions (20% O). The second bar depicts reduction in nitric oxide production or an attenuation of the invasion index of cells cultured under hypoxic condi nitric oxide effect. tions (1% O). The third bar depicts the invasion index of cells cultured under hypoxic conditions (1% O2) and admin SUMMARY OF THE INVENTION istered 10' M SNP. The fourth bar depicts the invasion index of cells cultured under hypoxic conditions (1% O) 0.011) An object of the present invention is to provide and administered 10' M GTN. The values indicated by “*” methods and formulations for administering low doses of were significantly different (p<0.05, n=6) using the Student nitric oxide mimetics to cells to inhibit and prevent a Newman-Keuls post-hoc test for pair-wise multiple com malignant cell phenotype. parison procedures. 0012. These methods and formulations are particularly 0018 FIG. 2 is a histogram showing the lung coloniza useful in controlling cancer by reducing its growth and tion ability of B16F10 mouse melanoma cells incubated for improving response to therapy. For example, methods and 12 hours in 1% or 20% O, in the presence or absence of formulations of the present invention can inhibit metastasis, 2x10 M GTN and injected i.v. (tail vein) into C57B16 invasiveness and progression of cells exhibiting a malignant female mice. Fourteen days later, mice were Sacrificed and phenotype. In addition, the methods and formulations can lungs were removed and fixed in Bouin’s fixative. Both induce or maintain dormancy of cells exhibiting a malignant melanotic and amelanotic metastatic colonies were counted phenotype at primary as well as Secondary Sites of tumors. under a dissecting microScope. The first bar depicts the Further, these methods and formulations can prevent or number of nodules observed in lungs of mice injected with decrease development of resistance of cells exhibiting a cells cultured in normal conditions (20% O). The second US 2005/0142217 A1 Jun. 30, 2005 bar depicts the number of nodules observed in lungs of mice 0024 Examples of cancers which can be treated via the injected with cells cultured in normal conditions (20% O) present invention include, but are not limited to, breast, and administered 2x10' M GTN. The third bar depicts the endometrial, uterine, ovarian, vaginal, cervical, colon, Stom number of nodules observed in lungs of mice injected with ach, esophageal, prostate, testicular, bone, skin, eye, head cells cultured in hypoxic conditions (1% O). The fourth bar and neck, brain, liver, pancreatic, renal, bladder, urethral, depicts the number of nodules observed in lungs of mice thyroid and lung cancer as well as leukemias, melanoma, injected with cells cultured in hypoxic conditions (1% O) lymphoma, and myeloma and Hodgkin's Disease. and administered 2x10' M GTN. 0.019 FIG. 3 shows circulating prostate specific antigen 0025 For purposes of the present invention, by “treat (PSA) levels in two patients, Patient A(FIG.3A) and Patient ment' or “treating” it is meant to encompass all means for B (FIG. 3B) who had undergone radical prostatectomy. controlling cancer by reducing growth of cells exhibiting a These patients were treated chronically with GTN adminis malignant cell phenotype and improving response to anti tered transdermally at a concentration of 0.03 mg/hour. A malignant therapeutic modalities. Thus, by “treatment' or sharp decline in plasma PSA levels was observed in both “treating” it is meant to inhibit the Survival and/or growth of patients within two months of administration of low dose cells exhibiting a malignant cell phenotype, prevent the NO mimetic therapy. In Patient A, this decline continued Survival and/or growth of cells exhibiting a malignant cell throughout the course of the study. In Patient B, further phenotype, decrease the invasiveness of cells exhibiting a increases in PSA levels were minimal. Plasma PSA levels malignant cell phenotype, decrease the progression of cells were measured using a radioimmunoassay that has an accu exhibiting a malignant cell phenotype, decrease the racy of +0.1 ng/ml. metastases of cells exhibiting a malignant cell phenotype, increase the regression of cells exhibiting a malignant cell 0020 FIG. 4 shows PSA levels in one patient with phenotype, and/or facilitate the killing of cells exhibiting a prostate cancer wherein the prostate is still intact. This malignant cell phenotype. “Treatment' or “treating” is also patient was administered three episodes of treatment, for meant to encompass maintenance of cells exhibiting a approximately one month each, of GTN, 0.03 mg/hour, 24 malignant cell phenotype in a dormant State at their primary hours a day. AS shown, following commencement of the first Site as well as Secondary sites. Further, by “treating” or two episodes, a decrease in the rate of increase in PSA levels “treatment' it is meant to increase the efficacy as well as was observed. Following commencement of the third epi prevent or decrease resistance to antimalignant therapeutic Sode, a decrease in PSA levels was observed. modalities. By “antimalignant therapeutic modalities it is 0021 FIG. 5 shows circulating PSA levels in one patient meant to include, but is not limited to, radiation therapies, with prostate cancer wherein the prostate is still intact. This thermal therapies, immunotherapies, , and patient was administered GTN chronically, transdermally at other therapies used by those of skill in the art in the a concentration of 0.03 mg/hour. Two months after chronic treatment of cancer and other malignancies. By “increasing GTN therapy was begun, the patient was administered the efficacy’ it is meant to include an increase in potency radiation therapy. AS shown, this combination therapy accel and/or activity of the antimalignant therapeutic modality erated the rate of PSA decrease to within three months. The and/or a decrease in the development of resistance to the expected average for a similar decrease in PSA levels antimalignant therapeutic modality. The present invention following radiation therapy alone is 12 months. also relates to methods of monitoring and/or diagnosing malignant cell phenotypes in an animal via measurement of DETAILED DESCRIPTION OF THE tumor Selective markers in an animal in the presence of low INVENTION dose NO mimetic therapy