Tumor therapy compounds that target extracellular hydroxyapatite

Abstract

A cation exchange nanoparticulate sulfonated polystyrene solution comprising a polymer, the polymer having at least one benzenesulfonate monomer and at least one Ca2+ calcium ion that is useful in the treatment of cancer.

Description

TUMOR THERAPY COMPOUNDS THAT TARGET EXTRACELLULAR HYDROXYAPATITEPrior Applications

[0001] This application claims benefit of US Serial Number 63 / 607,486, filed December 7, 2023, the contents of which are incorporated herein by reference. .Field of the Invention

[0002] Embodiments of the present invention include a novel injectable cation exchange nanoparticulate sulfonated polystyrene solution (NSPS), compounds of the present invention, and a monomer analog, for chelating calcium. Particularly, the present inventors have discovered compounds to chelate calcium from hydroxyapatite (HAP) lattice that is naturally produced by malignant tumors and deposited in their microenvironment biomarker. Thus, the novel NSPS and compounds of the present invention interact indirectly with the tumors by perturbing tumor microenvironment and changing it from acidosis state to alkalosis state which is deadly to tumor cells.

[0003] It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. The present inventors provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects.Background of the Invention

[0004] Hydroxyapatite (HAP), Caio(P04)60H2, once thought to be solely a ubiquitous component of bone and teeth, has also been shown to be produced by malignancies in cell cultures and in vivo. Formation of such tumor-associated HAP is postulated to involve alkalinephosphatase (ALP) mediated release of inorganic phosphate (Pi) that then combines with calcium to produce HAP crystals. These crystals are then deposited in the extracellular matrix and subsequently influence the tumor microenvironment (TME). TME-HAP has been shown to enhance tumor proliferation, progression, and migration by promoting mitogenesis and matrix metalloproteinase (MMPs) expression. MMPs promote tumor growth by degrading matrix barriers and by enhancing angiogenesis. The present inventors have shown in previous work that HAP -binding radiotracers such as FDA-approved 18F-labeled sodium fluoride (18F-NaF) and 99mTc-labeled methyl diphosphate (99mTc-MDP) could be used with positron emission tomography (PET) and single photon emission computed tomography (SPECT), respectively, to detect breast tumors, gastric tumors, and peritoneal ovarian tumors that contain HAP in the tumor microenvironment. Detection of tumor-associated HAP exhibited high specificity and high signal-to-background ratio (SBR) as HAP is absent in normal soft tissues. This strategy has subsequently been used by others for detecting primary breast tumors.

[0005] Triple negative breast cancer (TNBC), lacking estrogen, progesterone and HER2 receptors, is usually more aggressive, harder to treat, associated with chemoresistance, high recurrence rates, distant metastases, and poor overall survival (OS) compared to cancers that are hormone receptor and / or HER2 positive. Acidosis in the tumor microenvironment may play a role in alteration of drug structure and / or uptake, contributing to resistance to cytotoxic chemotherapy. Additionally, acidosis may result in elevated expression of programmed death ligand 1 (PD-L1), and diminished activity of cytotoxic T lymphocytes (CTLs). There is an urgent clinical need for new treatment paradigms that could improve the outcome for cancer patients with poor prognosis.

[0006] The present inventors have now successfully engineered a novel injectable cation exchange nanoparticulate sulfonated polystyrene solution (NSPS) designed to breakup TME- HAP and increase tumor extracellular pH (pHe) by chelating calcium, similar to cation exchange resins:2NSPS + Ca++NSPS2Ca + 2Na+.. ..(eq. 1) and in return releasing (PO4)- and OH- anions. The present inventors provide compelling evidence of the efficacy of NSPS in the treatment of tumors with TME-HAP in murine models.TME-HAP is absent from normal soft tissue but produced directly by tumor cells and is distinct from exogenously administered manufactured ultra-fine nano HAP (nHAP) injected directly into tumors or used as a vehicle for drug delivery.Summary of the Invention

[0019] In one aspect, the present disclosure provides compounds or compositions disclosed herein, and the pharmaceutically acceptable salts and solvates thereof, collectively referred to herein as “Compounds of the Disclosure” or “a compound of the present invention.”

[0020] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the present invention and one or more pharmaceutically acceptable carriers.

[0021] In another aspect, the present disclosure provides methods for treating a disease, disorder, or condition in a subject, comprising administering a therapeutically effective amount of a compound of the present invention to the subject.

[0022] In another aspect, the present disclosure provides methods for treating a disease, disorder, or condition in a subject, comprising administering a therapeutically effective amount of a compound of the present invention in combination with one or more optional therapeutic agents to the subject.

[0023] In another aspect, the present disclosure provides methods for treating a disease, disorder, or condition responsive to dissolving TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism, comprising administering a therapeutically effective amount of a compound of the present invention to a subject.

[0024] In another aspect, the present disclosure provides a pharmaceutical composition for treating a disease, disorder, or condition in a subject, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound of the present invention in a mixture with one or more pharmaceutically acceptable carriers.

[0025] In another aspect, the present disclosure provides a compound of the present invention for use in treating cancer in a subject in need thereof.

[0026] In another aspect, the present disclosure provides a compound of the present invention for use in the manufacture of a medicament for treating cancer in a mammal.

[0027] In another aspect, the present disclosure provides a therapeutic or prophylactic agent for cancer, which comprises a compound of the present invention.

[0028] Additional embodiments and advantages of the disclosure will be set forth, in part, in the description that follows, and will flow from the description, or can be learned by practice of the disclosure. The embodiments and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention as claimed.

[0007] The present inventors have developed a novel injectable cation exchange nanoparticulate sulfonated polystyrene solution (NSPS) that was engineered to dissolve TME- HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV- Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using18F-NaF for HAP and18F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCFlOa normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods.Brief Description of the Figures

[0008] Figure 1 shows the predicted structures of major components of NSPS. Analytical data (mass spectroscopy) along with FTIR and NMR suggest vinylbenzenesulfonate (CsHyOsS' [M-H] exact mass 183.0125) and hydroxylated vinylbenzenesulfonate (CsHyCUS- [M-H] exact mass 199.0074) as predominant components of NSPS.

[0009] Figure 2 shows the Structure of a novel NSPS monomer of the present invention. The structure and corresponding 1H NMR spectrum of the small molecule VU0945652, the active ingredient of NSPS.

[0010] Figure 3 shows that NSPS leads to tumor cell death in cells with HAP. Dead to total cell ratio of 4T1, MDA-MB-231 tumorigenic breast cell lines (both deposit HAP in their extracellular matrix), H292 lung tumor cell line which had no detectable ECM-HAP, and MCF10 normal breast cell line that lacks extracellular HAP. All cells treated with 7 pM NSPS and counted within 18-24 hours. * p<0.0001; t = 33.2, df =16; ** p<0.0001; t=23.7, df = 16; *** p = 0.0003, t = 4.6, df = 16; MCFlOa; p = 0.3464; t=0.97, df =16

[0011] Figure 4 shows that VU0945652, a compound of the present invention is the active ingredient of NSPS polymer and tumor cell death is dose dependent. (Left) Impact of 20 mg of VU0945652 on 4T1 breast and H292 lung tumor cultures. (Right) Dose effects of NSPS polymer (IX = 2.4 mg) and the small monomer VU0945652 (IX = 20 mg) on 4T1 breast cells cultured in osteogenic cocktail medium. Controls are cell cultures that received saline with culture media.1p < 0.0001; t=22.22, df=6; p< 0.0001; t = 9.99, df = 6; * p < 0.0001; t=31.01, df=6; ** p = 0.0025; t=5.000, df=6; *** p < 0.0001; t=18.42, df=6; **** p < 0.0001; t=12.25, df=6

[0012] Figures 5(A)-(D) show that NSPS induces acute alkalosis localized to the tumor. A-C are typical data from a single NSPS treated mouse bearing a 4T1 tumor in the mammary fat pad. (A) Sample T2 weighed MRI spectrum of a 4T1 breast tumor in the mammary fat pad of a white female Balb / c mouse. Regions of interest were drawn around the tumor. (B) CEST data acquired after injection of iohexol in the tumor (baseline or scan 0), just after i.v. injection of 25 mg / kg NSPS and every 7.25 min for a total of 10 scans post NSPS treatment. (C) Lorentzian fit of the tumor CEST data around the 4.3 ppm peak. (D) Changes in tumor extracellular pH compared to baseline (scan 0) is the average of 4 independent measurements per group (NSPS vs vehicle saline injected controls) on 4T1 tumor bearing mice. Data points displayed as means ± SE.

[0013] Figure 6 shows that NSPS increases tumor pH in vivo. Groups of mice bearing 4T1 tumors in the mammary fat pad received 25 mg / kg NSPS (i.v.), vehicle (saline), or nothing at all (shams); n = 3 per cohort. The tumors were then homogenized and the overall (macro) pH of tumors that received NSPS was significantly higher (p<0.05) than that of the other twogroups. * p = 0.0031, t =6.40, df =4; ** p = 0.0064, t = 5.23, df =4; sham vs saline: p = 0.8359, t = 0.22, df = 4.

[0014] Figures 7(A)-(N) show that NSPS dissolves TME-HAP and inhibits glucose metabolism for up to 1 week. Representative images of a MMTV-Neu breast tumors in the mammary fat pad of female FVP / n mice imaged with18F-NaF at (A) baseline (white arrow points to tumor) and (B) within 48 hours following i.v. injection of one-time 100 mg / kg of NSPS (see Table 1 for quantification and statistics). Approximately 24 hours following baseline18F- NaF imaging, the mice were imaged with18F-FDG PET at (C) baseline, (D) 24 hours post NSPS treatment and (E) 1 week post NSPS treatment. (F) Same mouse model imaged with FDG PET at baseline and (G) one week post vehicle (saline) injections. All tumors from all mice were harvested after the 1-week FDG scan and underwent IHC analyses. The tumors of the NSPS treated mice tested positive for cleaved caspase 3 (apoptosis) (H) throughout the tumors and negative for Ki 67 (proliferation) (J). Some HAP was observed in the tumor via von Kossa (K) and alizarin red S (L) staining. Black arrows point to positive (black) stains. This is consistent with18F-NaF uptake in the tumor following treatment with NSPS. The vehicle treated mice were completely negative for cleaved caspase 3 (M) and positive for Ki 67 (N).

[0015] Figures 8(A)-(G) show that NSPS has minimal impact on tumors lacking detectable extracellular HAP. Representative image of a xenograft model of H292 lung tumor imaged with (A)18F-NaF PET but the tumor was not detected with this radiotracer. White arrows point to where the tumor should be. (B) Same mouse imaged with18F-FDG PET at baseline and (C) 24 hours post treatment with 100 mg / kg NSPS. No changes in FDG uptake in the tumor were detected;18F-FDG uptake in the tumor at baseline and post NSPS was 2.3 ± 0.5 %ID / g in both cases. (D) Sections of harvested tumors which did not reveal positive cleaved caspase 3 but were positive for Ki 67 throughout the tumor (E). The tumors also tested negative for (F) von Kossa and (G) alizarin red S staining indicating absence of detectable TME-HAP. Thus, this H292 mouse model serves as an in vivo negative control for testing NSPS.

[0016] Figure 9 shows that onetime NSPS significantly inhibits tumor growth rate (tumor indolence). Growth curves of MDA-MB-231 xenograft breast tumors in female athymic nu / nu mice. Data were fitted with exponential growth curves (dashed lines). See text for fit parameters.

[0017] Figure 10 shows that NSPS has limited impact on skeletal bone. Three dimensional microCT images of femur bones harvested from age-matched white balb / c mice. Red streaks on bone are the pores. The mice received either 100 mg / kg one-time NSPS (left) or vehicle saline (right) ten days before harvesting. The bone specimen were imaged in a microCT at a nominal resolution of 6 pm.

[0018] Figures 11(A)-(B) show that there is no evidence of nephrotoxicity following treatment with NSPS. Mice imaged with (A) "mTc-MAG3 SPECT (for renal function) and (B) with CT following an i.p. injection of Optiray CT contrast The images were taken 24-48 hours after treatment with 100 mg / kg of NSPS. Normal renal function detected in both images.

[0019] Figure 12 shows thatNSPS is a polymer similar in characteristic to cation exchange resins. Spectrum of NSPS solution (top) and solute from NSPS (middle) analyzed by FT-IR spectroscopy at EAG laboratories. The spectra of each sample was compared to a spectral library and is most consistent with a sulfonated styrene compound (bottom spectrum) as expected for NSPS. Peak assignments are in data Table SI.

[0020] Figures 13(A)-(C) show 1H NMR Spectrum of NSPS prepared in D2O. NMR spectrum, analyzed by EAG laboratories (www.EAG.com), includes features consistent with the aliphatic protons on the polymer backbone (A), protons attached to both the aromatic and aliphatic backbone region (B), and the aromatic protons (C).

[0021] Figure 14 shows that the ultraviolet spectrum of NSPS consistent with polystyrene. Absorbance spectra of NSPS after dilution 10X or 100X into phosphate-buffered saline (PBS). At 10X dilution, the spectrum is dominated by scattering from the colloidal solution; at 100X, absorption features are evident at -228 nm and -255 nm consistent with the UV absorption spectrum of polystyrene.

[0022] Figure 15 shows dynamic light scatter (DLS) analysis of NSPS. Sample preparation which exhibited a z-average nanoparticulate diameter of 159 nm and poly dispersity index (PDI) of 0.224.

[0023] Figure 16 shows that NSPS shelf life is at least 1 year. Mass spectrum of NSPS stored in a refrigerator for one year (top) and two years (middle). While the 199 and 183 peakswere observed at 1 year, they were not present in the 2-year spectrum indicating that NSPS loses stability between 1 and years in storage.

[0024] Figure 17 shows that extracellular matrix HAP (ECM-HAP) is detectable in 4T1 breast cancer cells but not H292 lung cancer cells, in vitro. Staining with von Kossa and alizarin red S of 4T1 breast cancer cells grown on slides in DMEM median containing osteogenic cocktail for at least 11 days and H292 lung cancer cells in normal DMEM. Arrows point to example of positive stains. Sample MDA-MB-231 stained cells can be found in our previous work in reference 11.

[0025] Figure 18 shows little to no changes in pHe of controls or tumors lacking TME- HAP following treatment. T2 weighed MRI (left) and corresponding tumor CEST data (right) of a 4T1 breast tumor scanned at baseline and after vehicle (saline) injection (top panel) and H292 lung tumor following treatment with 25 mg / kg NSPS (bottom panel).

[0026] Figures 19 (A)-(J) show that NSPS inhibits glucose metabolism in many types of tumors with HAP, in vivo.18F-FDG PET images of: (A) nude mice with MDA-MB-231 breast tumors imaged at baseline (coronal view) and at (B) 24 hours and (C) 1 week post treatment NSPS; (D) 4T1 tumors in the mammary fat pad at baseline (sagittal view) and (E) 24 hrs post NSPS treatment; (F) PC3 prostate in nude mice at baseline (coronal view) and (G) 24 hrs post NSPS treatment; and (H) HCA-7 colon tumors in nude mice at baseline (axial view) and (J) 24 hrs post NSPS treatment. NSPS treatment for all mice was one-time 100 mg / kg / 0.2 ml injected i.v. A -75% reduction in FDG uptake was observed in all tumors following NSPS treatment. (Right panel; Top) IHC staining samples of cleaved caspase 3 and Ki 67 from harvested HCA 7 tumor section. Arrow points to example of positive stains. (Right panel; Bottom) Sample von Kossa and alizarin red S tumor sections. Arrows point to examples of positive stains. Proof of the presence of TME-HAP in MDA-MB-231 tumors was provided in previous work (see references 11 & 12).

[0027] Figures 20 (A)-(J) shows that NSPS is present in Tumor but absent in normal soft tissue. Mass spectroscopy of tumor and other organs harvested from white Balb / c mice with 4T1 tumors in the mammary fat pad and injected i.v. with either 100 mg / kg of NSPS where A) tumor at 10 min, B) tumor at 1 hr. C) Liver at 1 hr. D) Spleen at 1 hr. E) Kidneys at 1 hr. F) Bone at 1hr. G) Tumor at 4 hrs. and H) bone at 4 hrs or injected with vehicle (saline) where J) is harvested 4T1 tumor at 1 hr. NSPS was absent in soft tissue at all the time points tests but appeared to be present in Bone at least during the first hour while absent by 4 hours post NSPS injection.

[0028] Figure 21 shows that HAP is found in the microenvironment of HGSC. Clinical samples that was found to be either TME-HAPpos(Top panel) or TME-HAPneg(Bottom panel). Moving horizontally; the samples were stained with von Kossa for phosphates (left), alizarin red S for calcium (middle). Black spots are positive stains and indicate clusters of HAP at 40X magnification. (Right) Raman spectrum of the samples. A peak at -960 cm’1(where arrow points; yield = 2.6%) is consistent with the presence of HAP. Since the samples were placed on glass slides, the large peak between 990 and 1050 (yield = 100%) are silica interference. For TME-HAPnegtumors, yield at -960 cm’1< 0.1 %

[0029] Figure 22 shows that TME-HAP may be associated with reduced survival probabilities regardless of platinum chemotherapy responsiveness. The Kaplan-Meier probability survival curves display the survival probabilities of HGSC patients with tumors that were positive or negative for TME-HAP and were treated with platinum chemotherapy. The results suggest that TME-HAP positivity in ovarian cancer patients may be associated with poorer survival outcomes regardless of platinum chemo responsiveness.

[0030] Figure 23 shows thatNSPS treatment slows tumor progression. Left panel: Bioluminescence imaging was performed on mice bearing peritoneal ID8-luc tumors. Both mice were administered the same concentration of luciferin and imaged simultaneously at baseline (week 4). Subsequently, the mouse on the left was administered a one-time intravenous injection of 2.4 mg NSPS, while the mouse on the right received vehicle (PBS). Weekly imaging was performed on both mice simultaneously. Right panel: The luminosity curves of mice treated with NSPS (n=8) or PBS (n=7; controls) over several weeks. The results reveal that NSPS treatment led to a reduction in tumor growth, as evidenced by the lower luminosity levels in the NSPS- treated mice compared to the PBS-controls. The insert at the bottom provides a magnified view of the luminosity curves between weeks 5 and 6.

[0031] Figures 24 (A-J) showthatNSPS disrupts TME-HAP, inhibits tumor cell proliferation and promotes apoptosis in ovarian tumors. Representative18F-NaF PET images ofmice with peritoneal ID8-luc tumors imaged at (A) baseline (top) and 24 hours of i.v. administration of 2.4 mg NSPS (bottom), (B) baseline (top) and 24 hrs post PBS administration (control) (bottom). White arrows point to tumors at baseline for each mouse. (C) Representative anatomical CT image (0.11 mm nominal resolution) of the mouse in A. (D) BLI images of the mouse from A (left) and mouse from B (right) at baseline. (E) Post-mortem gross pathology. (F) von Kossa (top) and alizarin red S (bottom) staining for TME-HAP of the ID8-luc tumors from A. Black arrows point to positive stains. (G) Ki 67 stained sections of the NSPS treated mouse from A (top) and control mouse from B (bottom). (H) the same Ki 67 sections magnified to 20X. (J) Caspase 3 stained sections of the NSPS treated mouse from A (top) and the control mouse from B (bottom). Here, the purple background indicates viable cells while the brownish cleaved caspase 3 spots indicate apoptosis.

[0032] Figure 25 shows that onetime NSPS treatment mimics weekly cisplatin treatment. Time-activity curves (mean ± SD) of18F-NaF uptake (for TME-HAP) in the peritoneal cavity of mice injected with ID8-luc tumor cells and imaged weekly via PET starting at 4 weeks post intraperitoneal tumor cell injection. At the end of week 4 imaging session, the mice were treated with weekly injections of PBS (Group 1), 2 mg / kg cisplatin in PBS (Group 2), 2 mg / kg cisplatin for 2 weeks then switched to weekly PBS (Group 3; model of recurrence), or onetime 2.4 mg of NSPS (Group 4). SUV: standard uptake value, i.e., images were normalized to injected dose and mouse weight.

[0033] Figure 26 shows thatECM-HAP in culture. ID8-luc cells cultured on slides with osteogenic cocktail media for 21 days and then stained with von Kossa (left) and alizarin red S (right) confirming the presence of ECM-HAP. Arrows point to examples of positive stains.

[0034] Figures 27 (A-E) show characterization of a cisplatin-resistant derivative of ID8-luc ovarian cancer cells. ID8-luc cells were clonally selected following extended treatment with 10 pM cisplatin. (A) Response of parental ID8-luc (ID8-luc-PAR) and a cisplatin-resistant ID8-luc clone (ID8-luc-CP) to cisplatin was assessed by sulforhodamine B in vitro growth assay (72h treatment). Growth curves and IC50 values were generated using Prism software. ID8-luc PAR and ID8-luc CP cells were also injected IP into wild-type C57BL / 6 female mice. 30 days post injection (baseline), mice were given weekly injections of 2 mg / kg cisplatin intraperitoneally for21 days prior to sacrifice. ID8-luc CP cells maintained their relative resistance in vivo. (B-C) bioluminescence. (D) solid omental tumor implants. (E) ascites fluid and were measured. Values are mean ± SEM, n=5 per group, p values are Mann-Whitney test.Description of the Invention

[0035] As used in the specification, including attachments incorporated herein by reference, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

[0036] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0037] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat anexisting disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0038] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0039] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0040] The term “subject” refers to a target of administration. The subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0041] As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0042] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, “diagnosed with cancer” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition of the present invention or a compound or composition known to treat cancer.

[0043] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient tocause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0044] Accordingly, the compounds of the present invention may be used as single agents or in combination with one or more other drugs in the treatment, prevention, control, amelioration or reduction of risk of the aforementioned diseases, disorders and conditions for which the compounds of the present invention have utility, where the combination of drugs together are safer or more effective than either drug alone. As discussed above, the other drug(s) may be administered by a route and in an amount commonly used therefore, contemporaneously or sequentially with a disclosed compound. It is also envisioned that in some situatons, the combination of one or more active ingredients and a disclosed compound can be more efficacious than either as a single agent.

[0045] All publications mentioned herein, including those in the attachments incorporated herein by reference, are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date ofthe present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which need to be independently confirmed.

[0046] The nanoparticulate sulfonated polystyrene solution (NSPS) of the present invention: one example of the invention is a solution that comprises the monomer, named VU0945652 and polymer, nanoparticulate sulfonated polystyrene solution (NSPS) used for chelating calcium. This example was tested on cancer and found to have broad applicability in inducing tumor cell death, inhibiting tumor cell metabolism and growth.

[0047] VU0945652 represents at least one of a hydroxylated vinylbenzenesulfonate monomer and a vinylbenzenesulfonate monomer shown below:wherein x is 0-6, and is an attachment point for another monomer.

[0048] Accordingly, one embodiment of the present invention is a cation exchange nanoparticulate sulfonated polystyrene solution comprising a polymer, the polymer having at least one benzenesulfonate monomer and at least one Ca2+ calcium ion.

[0049] In one example, the monomers are bridged by a calcium ion, such as Ca2+.

[0050] In another example, the monomer is of the following formula:wherein n is an integer with a total value of from about 50 to about 300.

[0051] In another example, the monomer is of the following formula:wherein x is 0-6, andJVVU' is an attachment point for another monomer.

[0052] In yet another example, the mass of the monomer is about 199 Da ± 20 Da.

[0053] In another example, the molecular weight of the polymer is about 45-65 kDa, and up to about 800 kDa.

[0054] In another example, the number of monomer units is about 200-300.

[0055] In another example, the polymer has a calcium binding capacity of about 4.63 X I-22 mol per polymer molecule.

[0056] In another example, the average hydrodynamic diameter of the of the nanoparticulate is about 160nm ± about 20 nm, preferably about 160 nm ± about 6 nm.

[0057] In another example, the poly dispersity is about 0.1 - 0.5, preferably about 0.22 ± 0.02.

[0058] Salts, hydrates, and solvates of a compound of the present invention can also be used in the methods disclosed herein. The present disclosure further includes all possible stereoisomers and geometric isomers of a compound of the present invention to include both racemic compounds and optically active isomers. When a compound of the present invention is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent, for example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997). Resolution of the final product, an intermediate, or a starting material can be achieved by any suitable method known in the art. Additionally, in situations where tautomers of a compound ofthe present invention are possible, the present disclosure is intended to include all tautomeric forms of the compounds.

[0059] The present disclosure encompasses the preparation and use of salts of a compound of the present invention, including pharmaceutically acceptable salts. As used herein, the pharmaceutical "pharmaceutically acceptable salt" refers to salts or zwitterionic forms of a compound of the present invention. Salts of a compound of the present invention can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salts of a compound of the present invention can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Nonlimiting examples of salts of compounds of the present invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, tri chloroacetate, tri fluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedi sulfonate, benzene sulfonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference a compound of the present invention appearing herein is intended to include compounds of the present invention as well as pharmaceutically acceptable salts, hydrates, or solvates thereof.

[0060] The present disclosure encompasses the use of solvates of a compound of the present invention. Solvates typically do not significantly alter the physiological activity or toxicity of a compound, and as such may function as pharmacological equivalents. The term"solvate" as used herein is a combination, physical association and / or solvation of a compound of the present invention with a solvent molecule such as, e.g., a disolvate, monosolvate or hemisolvate, where the ratio of solvent molecule a compound of the present invention is about 2:1, about 1 : 1 or about 1 :2, respectively. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances, the solvate can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Thus, "solvate" encompasses both solution-phase and isolatable solvates. A compound of the present invention can be present as solvated forms with a pharmaceutically acceptable solvent, such as water, methanol, ethanol, and the like, and it is intended that the disclosure includes both solvated and unsolvated forms of a compound of the present invention. One type of solvate is a hydrate. A "hydrate" relates to a particular subgroup of solvates where the solvent molecule is water. Solvates typically can function as pharmacological equivalents. Preparation of solvates is known in the art. See, for example, M. Caira et al, J. Pharmaceut. Sci., 93(3 / 601-611 (2004), which describes the preparation of solvates of fluconazole with ethyl acetate and with water. Similar preparation of solvates, hemisolvates, hydrates, and the like are described by E C. van Tender et al., AAPS Pharm. Sci. Tech., 5(7 / Article 12 (2004), and A.L. Bingham et al., Chem. Commun. 603-604 (2001). A typical, non-limiting, process of preparing a solvate involves dissolving a compound of the present invention in a desired solvent (organic, water, or a mixture thereof) at temperatures above 20°C to about 25°C, then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by known methods, e.g., filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in a crystal of the solvate.

[0061] In another embodiment, a compound of the present invention is administered to a subject having cancer as a single chemotherapeutic agent.

[0062] In another embodiment, a compound of the present invention is administered to a subject having cancer in combination with one or more optional therapeutic agents. A compound of the present invention and optional therapeutic agent(s) can be administered in combination under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, a compound of the present invention is administered to the patient according to an intermittent dosing schedule.

[0063] In some embodiments, a compound of the present invention is administered prior to the optional therapeutic agent(s), e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks.

[0064] In some embodiments, a compound of the present invention is administered after the optional therapeutic agent(s), e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks.

[0065] In some embodiments, a compound of the present invention and the optional therapeutic agent(s) are administered concurrently but on different schedules, e.g., a compound of the present invention is administered daily while the optional therapeutic agent(s) is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, a compound of the present invention is administered once a day while the optional therapeutic agent(s) is administered once a week, once every two weeks, once every three weeks, or once every four weeks.

[0066] The therapeutic methods provided herein comprise administering a a compound of the present invention to a cancer patient in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typically, a compound of the present invention is administered in an amount from about 0.05 mg / kg to about 500 mg / kg, about 0.05 mg / kg to about 100 mg / kg, about 0.05 mg / kg to about 50 mg / kg, or about 0.05 mg / kg to about 10 mg / kg. The dosage of a composition can be at any dosage including, but not limited to, about 0.05 mg / week to about 100 mg / week. Particular doses include 0.05, 1, 2, 5, 10, 20, 500, and 100 mg / kg once daily, or once weekly. In one embodiment, a compound of the present invention is administered one, two, three, four, or five times a week, i.e., the compound of the present invention is administered according to an intermittent dosing schedule. These dosages are exemplary, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this disclosure. In practice, the physician determines the actual dosing regimen that is most suitable for an individual patient, which can vary with the age, weight, and response of the particular patient.

[0067] Therapeutically effective amounts of a compound of the present invention and optional therapeutic agent(s) can be formulated in accordance with standard pharmaceutical practices, are administered to a human subject in need thereof. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment (diagnosis) that takes into consideration signs, symptoms, and / or malfunctions that are present, the risks of developing particular signs, symptoms and / or malfunctions, and other factors.

[0068] Pharmaceutical compositions include those wherein a compound of the present invention and optional therapeutic agent(s) are administered in an effective amount to achieve its intended purpose. The exact formulation, route of administration, and dosage is determined by an individual physician in view of the diagnosed condition or disease. Dosage amount and interval can be adjusted individually to provide levels of a compound of the present invention and the optional therapeutic agent(s) that is sufficient to maintain therapeutic effects.

[0069] Toxicity and therapeutic efficacy of a compound of the present invention and optional therapeutic agent(s) can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the maximum tolerated dose (MTD) of a compound, which defines as the highest dose that causes no toxicity in a patient. The dose ratio between the maximum tolerated dose and therapeutic effects (e.g. inhibiting of tumor growth) is the therapeutic index. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0070] A therapeutically effective amount of a compound of the present invention and optional therapeutic agent(s) for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the subject, and ultimately is determined by the attendant physician. For example, dosage amounts and intervals can be adjusted individually to provide plasma levels of a compound of the present invention and / or optional therapeutic agent(s) that are sufficient to maintain the desired therapeutic effects. The desired dose conveniently can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example as one, two, three, four or moresubdoses per day. Multiple doses often are desired, or required. For example, a compound of the present invention can be administered at a frequency of: one dose per day; four doses delivered as one dose per day at four-day intervals (q4d x 4); four doses delivered as one dose per day at three-day intervals (q3d x 4); one dose delivered per day at five-day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5 / 2 / 5); or, any dose regimen determined to be appropriate for the circumstance.

[0071] The optional therapeutic agent(s) is administered in therapeutically effective amounts. For example, when the optional therapeutic agent(s) is an immune checkpoint inhibitor, and the immune checkpoint inhibitor is a monoclonal antibody, 1-20 mg / kg is administered as an intravenous infusion every 2-4 weeks. For example, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg and 2000 mg of the antibody may be administered.

[0072] Other embodiments of the present invention include methods of treating diseases where accumulation of calcium is a health risk (e.g. vascular calcification, soft tissue calcification, myositis ossificans... etc).

[0073] In one example of the present invention, within 24 hours of adding the small concentration of IX of NSPS (~7 pM), the present inventors observed significant tumor cell death (~ 10%, p<0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but <2% in H292 and MCFlOa cells that lack detectable HAP and in controls. Using CEST MRI, the present inventors found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration, in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in18F-NaF uptake post NSPS treatment as expected;18F‘ uptake in the tumor = 3.8 ± 0.5 %ID / g (percent of the injected dose per gram) at baseline compared to 1.8 ± 0.5 %ID / g following one-time treatment with 100 mg / kg NSPS. Of similar importance, is that18F- FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 hours of treatment with one-time NSPS which persisted for at least one week. Additionally, tumorgrowth was significantly slower (p<0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone.

[0074] Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

[0075] The present invention shows strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using the novel therapeutic of the present invention can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. It has been shown that TME- HAP is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue.

[0076] Embodiments of the present invention include an injectable cation exchange nanoparticulate sulfonated polystyrene solution (NSPS) dissolves TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and metabolism. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. Dissolution of TME-HAP using the novel NSPS of the present invention provides a new treatment paradigm for otherwise difficult and, in many times, fatal malignancies including triple negative breast cancer, either as an alternative or in combination with established therapeutics.

[0077] One example of a polymer of the present invention is shown below:

[0078] NSPS is a novel bridged nanoparticle suspension dominated by the hydroxylated vinylbenzenesulfonate monomer with a unique mass of 199 Da. The present inventors have shown that compounds of the present invention, as an active component of NSPS, functions to breakup HAP by chelating calcium (via wrap-around method) from the HAP lattice. The abundance of the released (PO4)'3and OH' anions and / or the potential formation of NaOH in the TME induce an acute alkalosis localized only to the TME. This perturbation of tumor pHe is localized and not systemically (whole body) and has not previously been described.

[0079] The present inventors show that a flat 2.4 mg / 0.2 ml of NSPS, or the equivalent of about 100 mg / kg, inhibits tumor glucose metabolism and growth. It is not uncommon to administer a flat dosage of nanoparticles rather than a dose calculated in mg / kg. A pilot study at 0.6 mg or the equivalent of ~25 mg / kg had a modest but not significant (p>0.05) impact on tumor glucose metabolism as assessed using18F-FDG PET imaging. Generally, polymer-based drugs are administered at high doses to achieve therapeutic efficacy due to the intrinsic physicochemical properties that may limit circulation time in the blood plasma and / or poor aqueous solubility which reduces bioavailability. On the other hand, small molecule drugs are distributed not only in tumor tissue, but are also widely distributed in all healthy tissues, which may result in adverse side effects. This is one of the advantages of polymeric macromoleculardrug formulations (like NSPS) which are likely to spare healthy tissue from toxicity by preferentially accumulating within tumor microenvironment through the enhanced permeation and retention (EPR) effect that appears mediated by disorganized tumor vasculature and poor lymphatic drainage of tumors. The mass spectroscopy biodistribution results confirm the presence of NSPS in the tumor. Meanwhile, absence of EPR and large vasculature to bone surface may explain absence of adverse effects of NSPS to bone. Without being bound by theory or mechanism, it is believed that the compact structure of HAP on bone surface, large skeletal surface area, limited vascularity of bone surface, and continuous bone remodeling may contribute to the limited interaction of NSPS with bone. By contrast, tumor HAP appears diffusely scattered within the extracellular matrix, which combined with the large and disorganized tumor vasculature, affording susceptibility to calcium chelation and dissolution. Thus, there is an advantage to having a large size HAP dissolving agent such that it does not penetrate bone and is specific targeting to TME-HAP. Furthermore, though trabecular bone CT parameters (BV / TV, Tb.Th, Tb.N, and Tb.TMD, see table 3) of the treated mice appeared slightly lower, no significant differences were found in the overall bone microCT data between mice treated with NSPS and vehicle suggesting that NSPS at 100 mg / kg had minimal adverse effects on bone.Table 3: NSPS has minimal impact on bone growth. Toxicity measurements on distal femur (trabecular bone) harvested from female white balb / c mice 10 days following injection of one-time 100 mg / kg NSPS or vehicle (saline). Data presented as mean ± SD (n = 10 per group), p > 0.05 between the two groups for any measurement.

[0080] While 10% cell death may appear modest, in vitro, for cells containing ECM-HAP, in the context of treatment with nanoparticles, 10% efficacy within 18 hours is relatively high.Polymer-based drugs typically take days and higher concentrations, e g., mM, to achieve similar efficacy.

[0081] There are FDA approved cation exchange sulfonated polystyrenes such as Kayexalate with high affinity for potassium used for patients with hyperkalemia with dosage as high as 800 mg / kg. Kayexalate is a linear polymer and has negligible affinity for calcium. As expected, it had no therapeutic effects on our tumor models even at the maximum dose dissolved in 0.2 ml saline and administered once per hour at a rate of 0.05 ml each hour as the present inventors did with NSPS.

[0082] Dissolution of TME-HAP with NSPS had no perturbatory impact on blood concentrations of calcium and phosphates. These results indicate that NSPS treatment did not cause systemic effects, e.g., release of calcium or phosphate from bone as no evidence of significant interaction of NSPS with bone was found. Although a localized response was evident in the tumor microenvironment via the18F-NaF,18F-FDG, and IHC analyses, the released calcium and phosphate following dissolution of TME-HAP may be too modest to cause a detectible change in the concentrations of those molecules in the blood. These ions may be absorbed back into the tumors cells from the microenvironment via transcellular and paracellular pathways, and / or they may be absorbed by bone remodeling.

[0083] Following treatment with 100 mg / kg of NSPS, tumor glucose metabolism was inhibited for at least one week which may indicate an irreversible change in the tumor metabolic activity. Tumor acidity is one of the hallmarks of cancer and likely contributes to metabolic reprogramming. Of similar importance is that metabolic activity of normal tissue was uninterrupted by NSPS making dissolution of TME-HAP an attractive cancer therapeutic target that, until now, has not been investigated. Tumor microenvironment is generally more acidic than the surrounding normal tissue. A sudden and rapid switch from acidosis to alkalosis may be toxic to tumors. Modulation of the formulation of NSPS to reduce the nanoparticle size may further enhance efficacy.

[0084] The present inventors could not obtain evidence of TME-HAP with H292 although the present inventors detected a modest impact of NSPS and VU0945652 on H292 in culture. Without being bound by theory or mechanism, this may indicate that TME-HAP may be associated with this cell line but is undetectable by von Kossa and alizarin red S even at 40x magnification. However, these data may indicate that the threshold of HAP abundance in theextracellular matrix needed to perturb the tumor microenvironment with NSPS, may be very small.

[0085] While no formal behavioral studies were carried out, no changes in the overall behavior, food, or water consumption in any of the NSPS-treated mice were observed. Further, none of the NSPS treated mice exhibited cell lysis syndrome even within mice bearing large tumors (e.g. PC3). Cell lysis can occur with current chemotherapeutics. While some increased apoptosis (-10% increase) and reduced Ki 67(~10% reduction) was detected in the tumors at 24 hours post treatment with NSPS compared to controls (see Fig 19), apoptosis was detected throughout the tumors by 1 week post NSPS treatment (see Fig 7).18F-FDG uptake was reduced by >75% in the tumors with HAP within 24 hours post NSPS treatment.

[0086] The present invention provides compelling evidence that NSPS treatment is efficacious in tumors with extracellular HAP with minimal impact on tissues that do not have HAP such as normal soft tissue or tumors lacking HAP.

[0087] The advantages of the cancer treatment approach of the present invention include: (i) Ubiquity, any tumor with TME-HAP is most likely to respond well to NSPS regardless of the tumor type or stage; (ii) Response time, pharmacodynamic parameters such as inhibition of tumor glucose metabolism, reduced proliferation, and increased tumor cell apoptosis can be observed within 24 hrs after administration of NSPS verifying efficacy, and (iii) Lack of systemic toxicity, the present inventors found no evident NSPS toxicity in normal tissue or skeletal bone.

[0088] Enhanced efficacy of NSPS at lower pH and for tumors that are more sensitive to pHe changes is anticipated. However, NSPS treatment has limited efficacy in tumors that lack detectable TME-HAP, e.g., H292 lung tumor cells, and that may also include tumor heterogeneity in which HAP is not distributed throughout the tumor. Cox et al. and Wen et al. have demonstrated that tumorigenic cells produce and deposit HAP in cell culture when the culture medium contained ascorbic acid and P-glycerophosphate (PG). The production of tumor associated HAP, in vivo, therefore, may be dependent on a number of variables including distance to vasculature, dietary factors (e.g., vitamins C & D), and calcium regulation and concentration in the blood. Additionally, Han et al. have demonstrated that repeated injectionsof nanoparticulate HAP (nHAP) exogenously can reach tumors and indirectly results in endocytosis and inhibition of tumor cells at large doses. Though not the same as TME-HAP, it may be possible that pretreatment of patients with nHAP for a few days to deliver HAP to tumors that lack HAP, or increased HAP abundance for tumors already containing HAP, followed by NSPS to induce alkalosis localized to the tumor would potentially maximize tumor efficacy. The disadvantage of such a dual therapeutic approach is that nHAP will also reach normal healthy soft tissue. Nonetheless, this dual therapy approach would broaden the application of NSPS to tumors like H292 lung that lack detectable TME-HAP. Other potential ways to maximize tumor efficacy would be to pretreat tumors with NSPS to inhibit tumor glucose metabolism followed by conventional chemo and / or immunotherapeutics (perhaps at reduced doses) and minimize adverse effects of those therapeutics.

[0089] It is important to distinguish between the inventive approach using NSPS to dissolve TME-HAP to induce an acute localized alkalosis in the tumor microenvironment from other work that aimed at neutralizing tumor microenvironment using methods with (i) systemic sequelae, e.g., treating with bicarbonates, (ii) inhibition of specific receptors that are also present in normal soft tissue, or (iii) targeting intracellular metabolism so as to reduce the intracellular pH and viability of tumor cells but without discrimination from normal soft tissue. While other prior strategies had efficacy in reducing tumor growth, confounding systemic effects were also evident, e.g., changes in whole body pH and metabolism and attendant severe adverse effects such as extreme fatigue, seizures, and irreversible normal tissue damage limiting clinical potential. Therefore, the strategy of the present invention is distinct and designed to target TME- HAP to temporarily elevate tumor pH locally without changing whole body pH. As HAP is generally absent in normal soft tissue and there are no other known injectable compounds that can breakup TME-HAP in vivo, NSPS would be a one-of-a-kind and first in a class of novel cancer therapeutics. NSPS could also potentially be used in the treatment other diseases in which HAP is expressed in the extracellular matrix, e.g., hepatic metastases in the clinic or for detecting chronic tuberculosis in mice.

[0090] Thus, the present inventors have shown that one-time treatment with NSPS was efficacious in treating tumors with TME-HAP as measured by reduced TME-HAP mineralization, abrogation of tumor metabolic activity, and inhibition of tumor growth withminimal adverse effects. Attacking solid tumors by altering the pH of the TME only and not systemically by dissolution of TME-HAP using NSPS is a novel approach to therapy. Dissolution of TME-HAP using NSPS to elevate tumor pHe locally without changing whole body pH is an attractive approach to anti-cancer therapy. NSPS has significant potential to be a paradigm changing approach to the treatment of cancer patients with poor prognosis.

[0091] Additional, nonlimiting, exemplary chemotherapeutic compounds, one or more of which may be used in combination with compounds of the present invention include: avastin, daunorubicin, adriamycin, Ara-C, VP- 16, teniposide, mitoxantrone, idarubicin, carboplatinum, PKC412, 6-mercaptopurine (6-MP), fludarabine phosphate, octreotide, SOM230, FTY720, 6- thioguanine, cladribine, 6-mercaptopurine, pentostatin, hydroxyurea, 2-hydroxy-lH-isoindole-l,3- dione derivatives, l-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof, l-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate, angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474, SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610, bevacizumab, porfimer sodium, anecortave, triamcinolone, hydrocortisone, 11-a-epihydrocotisol, cortex olone, 17a-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone, dexamethasone, fluocinolone, a plant alkaloid, a hormonal compound and / or antagonist, a biological response modifier, such as a lymphokine or interferon, an antisense oligonucleotide or oligonucleotide derivative, shRNA, and siRNA.

[0092] A number of suitable optional therapeutic, e.g., anticancer, agents are contemplated for use in the therapeutic methods provided herein. Indeed, the methods provided herein can include, but are not limited to, administration of numerous optional therapeutic agents such as: agents that induce apoptosis; polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimetics (e.g., gossypol or BH3 mimetics); agents that bind (e.g., oligomerize or complex) with a Bcl-2 family protein such as Bax; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-a) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); genetherapy reagents e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like. Numerous other examples of optional therapeutic agents such as chemotherapeutic compounds and anti cancer therapies suitable for co-admini strati on with the disclosed compounds are known to those skilled in the art.

[0093] In certain embodiments, anticancer agents comprise agents that induce or stimulate apoptosis. Agents that induce or stimulate apoptosis include, for example, agents that interact with or modify DNA, such as by intercalating, cross-linking, alkylating, or otherwise damaging or chemically modifying DNA. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g, TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (e.g. , epidermal growth factor receptor (EGFR) kinase inhibitor. Additional anticancer agents include: vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g, HERCEPTIN, RITUXAN, ZEVALIN, and A VASTIN); antiestrogens (e. , raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g, celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g, irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.

[0094] In still other embodiments, the therapeutic methods provided herein include administering to a subject having cancer (a cancer patient) therapeutically effective amounts of a compound of the present invention, and at least one additional optional therapeutic agent, e.g., ananti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and / or animal derived compounds).

[0095]

[0096]

[0097]

[0098] In still further embodiments, chemotherapeutic agents suitable for use in the methods of the present disclosure include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g, L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g, hydroxyurea); 9) methylhydrazine derivatives (e.g, procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (e.g., mitotane (o,p - DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., di ethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g, testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g, flutamide): and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).

[0099] Any oncolytic agent that is routinely used in a cancer therapy context finds use in the therapeutic methods of the present disclosure. For example, the U.S. Food and Drug Administration (FDA) maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the FDA maintain similar formularies. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.

[0100] Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG- 013736, AGROIOO, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin- 1 , buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, efl ornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hul4.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin- 12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab, mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafm, MS- 275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS-9, O6-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpimase, rebeccamycin analogue, rhu Angiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-l, S- 8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifamib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.

[0101] In one embodiment, the optional therapeutic agent comprises one of the anti-cancer drugs or anti-cancer drug combinations listed in the following table.[00102J For a more detailed description of anticancer agents and other optional therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al. 2002.

[0103] In some embodiments, methods provided herein comprise administering a compound of the present invention in combination with radiation therapy. The methods provided herein are not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to a patient. For example, the patient may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, andcombinations thereof. In some embodiments, the radiation is delivered to the patient using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.

[0104] The source of radiation can be external or internal to the patient. External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by patients. Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g, using particles attached to cancer cell binding ligands).Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.Examples

[0105] Example 1: Methods

[0106] Preparation of NSPS.

[0107] Six hundred milliliters (600 ml) of deionized water were added to 600 g of Amberlite IR120 Na+(Sigma Aldrich) beads + 6 mL of 0.9% clinical grade saline (Vanderbilt University Medical Center, VUMC, Pharmacy); i.e. 1 : 1 :0.01 mixture. The mix was stirred at 650 rotations per minute for at least 5 days or until the mixture had a milk-like white appearance. Then, the mixture was filtered through a 0.2 micrometer membrane (VWR, Suwanee GA USA) and allowed to air dry in a bio-hood. The dried resolute which weighed 2.4 ± 0.3 mg per 5 mL filtered mixture, was then mixed with 0.02 ml biograde ethanol (96% bio-EtOH) (Sigma Aldrich) and 0.2 ml saline. This final formulation is refered to as a nanoparticulate sulfonated polystyrene solution (NSPS) which was used for characterization and preclinical tests.

[0108] Properties of NSPS.

[0109] Triplicate independent samples of NSPS were analyzed by:

[0110] Fourier Transform Infrared Spectra (FT-IR) and nuclear magnetic resonance (NMR) spectra were carried out by EAG Laboratories (Maryland Heights, MS) on triplicate preparations of NSPS and the solute following evaporation of the water; NMR spectral analysis was also confirmed by the Vanderbilt Small Molecule NMR Facility Core (Vanderbilt University School of Medicine, Nashville, TN).

[0111] Mass spectrometry: high resolution mass spectrometry was performed in the Vanderbilt Mass Spectrometry Core Facility. Samples were analyzed by direct liquid infusion using an Orbitrap mass spectrometer (Thermo-Finnigan, San Jose, CA) equipped with an Ion- Max source housing and a standard electrospray (ESI) ionization probe in positive and negative ion modes at a resolving power of 60,000 (at m / z 400).

[0112] Particle size and distribution parameters were measured by dynamic light scattering (DLS) using a DLS Malvern Nano ZS instrument (Malvern Instruments, United Kingdom) in the Vanderbilt Institute of Nanoscale Science and Engineering (ViNSE). This step was repeated in triplicates 3 times on 3 different preparations of NSPS, i.e. Ntotai = 9.

[0113] Ultraviolet absorption (UV) spectroscopy: UV absorbance spectra were obtained using a UV-2501PC dual -beam spectrophotometer (Shimadzu) with 10 mm pathlength quartz cuvettes.

[0114] Based on the results, the present inventors identified a novel structure that was shown to be the cytotoxic active ingredient in NSPS. Therefore, the present inventors synthesized a novel sulfonated benzene monomer (VU0945652) with mass of 199 Daltons at the Vanderbilt Chemical Synthesis Core from 3 -bromobenzenesulfonic acid and potassium vinyltrifluoroborate using palladium cross-coupling reaction.

[0115] NSPS impact on calcium phosphates phantoms.

[0116] To confirm the theory that NSPS increases pH by dissolution of TME-HAP, the present inventors filled phantoms with 10 ml of deionized water + 1 mg of calcium phosphates salt (Thermofisher) + either 0.2 ml of lx NSPS (i.e. concentration of NSPS = 4.27 nmol / ml for 55 kDa molecular weight) or vehicle (0.2 ml saline). The pH of the phantoms was measuredusing a standard pH meter (Vernier Go Direct, OR, USA) at baseline and at 24 hrs and 7 days post NSPS / vehicle addition.

[0117] Assessing the In vitro efficacy of NSPS.

[0118] NSPS efficacy was tested using two tumorigenic breast cell lines that produce extracellular matrix HAP (ECM-HAP); mouse 4T1 and MDA-MB-231. In addition, MCFlOa normal mammary epithelial cell line that does not produce detectable ECM-HAP were used. The present inventors found that H292 lung tumors in xenograft models and in cell culture lacked detectable TME-HAP or ECM-HAP. Therefore, H292 tumors serve as a negative control, in vivo, while the MCFlOa cell line serves as a suitable negative control in vitro. Cells were cultured in plates using 10% FBS-supplemented DMEM glucose rich medium or RPMI-1640 (for H292) with 1% penicillin / streptomycin. Additionally, the 4T1 culture contained an osteogenic cocktail consistent of 50 pgmU1ascorbic acid and lOpM P-glycerophosphate (PG) as recommended by Cox and others for at least 11 days. Cells grown in osteogenic cocktail exhibited changes in morphology consistent with those reported by Morgan and colleagues. When each of the cell lines reached -70% confluency, they were split into 24 wells (1 ml per well).

[0119] NSPS (X = 2.4 mg in 0.02 ml Bio-EtOH + 0.2 ml saline) was diluted with 6 ml of the culture medium, i.e., concentration of NSPS = 7.02 nmol / ml (-7 pM) for 55 kDa molecular weight. For controls, vehicle (0.2 ml saline + 0.02 ml bio-EtOH) were mixed with 6 ml of the culture media. When all cells reached -70% confluency in the 24 wells, the original media was aspirated and then added 1 ml of the media + NSPS or vehicle. About 18 hours later, the number of total cells and dead cells were measured using a hemocytometer and trypan blue. The test was repeated on the 4T1 cultures at 0.3 X concentration of NSPS.

[0120] To verify that active ingredient in NSPS is VU0945652, the present inventors repeated the above study except this time, X = 20 mg of VU0945652 were mixed with 0.02 ml bio-EtOH + 0.2 ml saline with 6 ml culture media (16.2 pmol / ml). Then the present inventors added 1 ml of VU0945652 + media or vehicle + media to each well in a 24 well plate containing 4T1 breast cultured in osteogenic media or H292 lung cultured in RPM1400. The test was repeated on the 4T1 cultures at 0.3 X concentration of VU0945652.

[0121] Statistical comparisons between treatment and control (vehicle) cell cultures were conducted using unpaired two-tailed t-tests for each treatment type (NSPS, VU0945652), each treatment dose, and each cell line

[0122] In vivo studies with NSPS.

[0123] Mouse models.

[0124] All animal studies were approved by the Vanderbilt University Institutional Animal Care and Use Committee (IACUC). The present inventors used the following mouse models for studying the mechanisms of action and efficacy of NSPS:

[0125] l-MDA-MB-231 human breast cells (IxlO6) were injected in the flank of immunocompromised female athymic nu / nu mice (8 wks; n = 12) similar to previous work.

[0126] 2- 4T1 mouse breast cells (5xl05) were injected in the mammary fat pad of immunocompetent female Balbc / 6 mice (8 wks; n = 10) under the 4thnipple.

[0127] 3-MMTV-Neu breast cells (IxlO6) were surgically placed on the mammary fat pad of immunocompetent syngeneic FVB / n mice (8 wks; n = 7) under the 4thnipple similar to previous work.

[0128] 4 -H292 human lung cells (5xl06) were injected in the flank of immunocompromised female athymic nu / nu mice (8 wks; n = 8).

[0129] 5 -PC3 human prostate cells (5xl06) were injected in the flank of immunocompromised male athymic nu / nu mice (8 wks; n = 8).

[0130] 6-HCA-7 colon cells (IxlO6) were injected in the flank of immunocompromised male athymic nu / nu mice (8 wks; n = 8).

[0131] Studies were commenced when tumor size was at least 200 mm3as measured by calipers. The length of time after engraftment to achieve this size depended on the model and ranged from 2 weeks (e.g. 4T1) to approximately 10 weeks (e.g. H292).

[0132] Tumor extracellular pH (pHe) measurement, in vivo.

[0133] All animal procedures were approved by the inventors’ Institution’s Animal Care and Usage Committee.

[0134] For in vivo assessment of tumor pHe, 4T1 breast or H292 lung tumor bearing mice (n = 4 per group; NSPS vs vehicle saline) described above were imaged via magnetic resonance imaging (MRI) at 7 Tesla using the chemical exchange saturation transfer (CEST) approach, an established method for measuring extracellular pH in vivo. Clinical grade iohexol; 300 mg / ml Iodine (300 mgVml Omnipaque, GE Healthcare, purchased from VUMC pharmacy) was injected (0.2 ml) intraperitoneally and another 0.2 ml were injected directly into the tumors similar to Chen et al. The mice were scanned at baseline and immediately following an intravenous injection, via jugular vein catheter, of either a test dose of 25 mg / kg (0.6 mg) NSPS in 0.05ml saline or vehicle (0.05 ml saline). The doses were administered after a 0.05 ml saline pre-flush and followed by an additional 0.05 ml saline flush post NSPS or vehicle injection. The mice were then scanned up to 9 more times using the same CEST MRI protocol. Details of the CEST MRI protocol are provided. Each scan, whether baseline or post NSPS / veh injection, lasted 7.25 min. The injection of NSPS / veh took less than 60 seconds before the post treatment CEST scans started. The raw CEST data were fitted with a Lorentzian curve and compared to phantoms using the same MRI protocol. Details of the Lorentzian fitting approach are outlined in the data. The phantoms were prepared by mixing 0.2 ml of Omnipaque 300 with 1 mL deionized H2O and NaOH or HCL was added in 0.005 ml aliquots to adjust the pH. Seven phantoms with pH ranging between 6.15 and 9.15 were then scanned with the same CEST MRI protocol. Since iohexol has only one peak at ~ 4 ppm making it difficult to obtain absolute quantification of pH, the present inventors used the phantom as a map to determine the direction of change of pH in vivo.

[0135] For ex vivo pH analysis, groups of 4T1 orthotopic tumor bearing mice (n = 3 per group) were treated with 25 mg / kg / 0.05ml NSPS, i.v., 0.05 ml vehicle (saline), or no treatment. Within 20-30 min, the tumors were harvested and homogenized. The pH of the homogenates were measured directly using a Verner pH meter. The meter was thoroughly washed in deionized water and wiped clean between each reading.

[0136] In vivo efficacy of NSPS.

[0137] MMTV-Neu breast tumor mice received an intravenous (i.v.) injection of ~18 MBq of18F-NaF and were imaged 1 hour later for 20 min in an Inveon microPET / CT (Siemens preclinical, Knoxville TN). Approximately 24 hrs later, the mice received an i.v. injection of 18 MBq of18F-FDG and were imaged 40 min later for 20 min via PET. At the end of the scan and based on the pHe studies where pHe began decreasing after one hour, the mice received an i.v. injection of 25 mg / kg (0.6 mg / 0.05ml saline) of NSPS every 60 ± 15 min four times for a total treatment dose of 100 mg / kg (2.4 mg) NSPS in 0.2 ml solution (n = 4), which the present inventors refer to as one-time 100 mg / kg NSPS, or vehicle (0.05 ml saline) every 1 hr four times (n = 3). Within 18 hours of the final NSPS / vehicle injection, the mice received ~18 MBq of18F- FDG again and were imaged 40 min later via PET for 20 min. The next day, the mice received ~ 18 MBq of18F-NaF and were imaged 60 min later via PET for 20 min. All data sets were reconstructed using the three-dimensional (3D) ordered subset expectation maximization / maximum a posteriori (OSEM3D / MAP) algorithm into 128 x 128 x 95 slices with a voxel size of 0.095 x 0.095 x 0.08 cm3at a beta value of 0.01. The PET images were normalized to the injected dose. Three dimensional regions-of-interest (RO Is) were drawn around the tumor and skeletal bone in the18F-NaF PET images and around the tumor, liver, heart, brain, kidneys, lungs, and muscle in the18F-FDG PET images. The radiotracer uptake in each ROI were compared between the post NSPS / veh administration and baseline. Based on the results, the18F-FDG PET scans were repeated 1 week later using the same protocol. Then the mice were euthanized and tumors were harvested. Paraformaldehyde-fixed paraffin embedded sections were analyzed by immunohistochemistry for Ki 67 (proliferation) and cleaved caspase 3 (apoptosis).

[0138] The studies were repeated on MDA-MB-231 breast (n = 6 per group), 4T1 breast (n = 5 per group), PC3 (n = 4 per group) prostate, HCA7 (n = 4 per group) colon, and H292 lung (n = 4 per group) tumor mouse models. The H292 mouse model was used as negative controls due to the absence of detectable TME-HAP and absence of18F-NaF uptake in the tumor (please see results).

[0139] Tumor growth studies.

[0140] A separate tumor growth study was conducted on 4T1 tumor bearing mice that received either one-time 100 mg / kg of NSPS (divided into 4 equal injections every ~1 hour as described above) or saline (n = 10 per group). Tumor size was measured using calipers at 1 and 3 wks post NSPS / saline injection.

[0141] Biodistribution of NSPS and toxicity studies.

[0142] Mass spectroscopy of tissue samples. Balb / c mice bearing orthotopic 4T1 breast tumors (-400 mm3) received i.v. injections of 25 mg / kg of NSPS or vehicle (saline). Then the mice were euthanized at the following time points (n =3 per time point): 10 min, 1 and 4 hours post NSPS administration. Tumor, blood, liver, spleen, bone, lung, heart, kidneys, brain, and muscle were harvested and flash frozen at -80 °C. On the day of the analyses, the tissue samples were thawed and mixed with acetonitrile (1 :1) and homogenized then centrifuged. The supernatants were extracted and subjected to mass spectroscopy in negative ion mode using the same setup parameters used to study the properties of NSPS described above.

[0143] Bone toxicity studies. Bone mineralization density was evaluated on the midshaft and metaphysis of femora, expected to be vulnerable to HAP depletion, harvested from similar aged (12 weeks old) litter white female balb / c mice 10 days following i.v. treatment with either one-time 100 mg / kg NSPS (n = 10) or vehicle (saline; n = 10). After aligning the long axis of the bone with the specimen tube, each femur mid-diaphysis (1.848 mm in length) and the distal femur metaphysis (3.000 mm in length) were imaged using the following scan parameters (Scanco Medical pCT50 scanner, Briittisellen, Switzerland): X-ray tube voltage of 70 kVp drawing 114 pA, an isotropic voxel size of 6 pm, and an acquisition of 2000 projections per 360° rotation with an integration time of 300 ms. Weekly HAP phantom scans enabled conversion of attenuation values to bone mineral density following reconstruction. After contours were fit to the periosteal surface and endosteal surface (femur mid-diaphysis) or inserted several voxels from the endosteal surface (distal femur metaphysis) per previously published studies, the present inventors applied Scanco evaluation scripts to each region of interest to determine cortical bone and trabecular bone parameters. The global threshold was >828.1 mgHA / cm3(Gaussian image noise filter: sigma = 0.2 and support = 1) and >417.2 mgHA / cm3(sigma = 0.8 and support = 1) for the mid-diaphysis and metaphysis, respectively. These scripts provided thefollowing: cortical tissue mineral density (Ct.TMD), cortical thickness (Ct.Th), polar moment of inertia (J), resistance to bending on minor axis (Cmin), cortical bone area (Ct. Ar), trabecular tissue mineral density (Tb.TMD), trabecular bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th). Cortical porosity (Ct.Po), pore number, pore thickness, and pore spacing were also determined by applying a global, inverse threshold of <1021.3 mgHA / cm3 (sigma = 0.3 and support = 1) and evaluating the segmented image with an algorithm similar to the one for trabecular bone.

[0144] Plasma calcium and phosphate levels. Female Balb / c mice bearing 4T1 tumors -500 mm3in size, as measured by calipers, were treated with either one time 100 mg / kg NSPS or saline injected i.v. (n = 9 per group). Approximately twenty-four hours later, the mice were euthanized and blood was immediately collected via cardiac puncture. The blood was centrifuged and calcium and phosphate level were measured in the plasma using colorimetric assay kits (Abeam); ab 102505 for calcium and ab65622 kit for phosphate levels according to the manufacturer’s protocol. Briefly, 10 pl plasma was diluted in 40 pl deionized water (dFFO) in 96 well plates. Then 60 pl of calcium buffer and 90 pl of colorimetric reagent were added to each well and allowed to incubate at room temperature for 5-10 min. The wells were then scanned in a Gen 5.0 microplate reader (BioTek Industries, VT) in absorbance mode with a 575 ± 5 nm fdter. For phosphate concentration, 20 pl plasma was diluted in 2.98 ml dFEO. Then 0.2 ml of the diluted samples were placed in 96 wells and 30 pl of the phosphate reagent was added to each well and allowed to incubate at room temperature for 30 min. Then the plates were scanned in a microplate reader with an absorbance filter of 650 ± 5 nm filter.

[0145] Onco-nephrology evaluation. Nephrotoxicity is a common complication of many chemotherapeutics. Therefore, the present inventors carried out onco-nephrology to assess renal function as follows: 4T1 bearing mice were treated with one-time 100 mg / kg NSPS. -37 MBq of99mTc-MAG3 were injected i.v. in 4T1 bearing mice and with SPECT imaging within 48 hours after NSPS administration.

[0146] Example 2: Results

[0147] Properties of NSPS.

[0148] Triplicate independent analyses of NSPS samples resulted in the following findings:

[0149] Fourier Transform Infrared Spectra (FT-IR) and nuclear magnetic resonance (NMR) confirmed that NSPS was consistent with sulfonated polystyrene. Additionally, evaporation of water from the mixture yielded solutes with measured characteristics similar to Amberlite IR 120 Na+beads and P-NaSS (Ps-100), see Figs 12, 13 and Table SI, below.

[0150] Table S 1: FT-IR Results for NSPS saturated solution and solute. The peaks are shown in figure 15 and are consistent with those of polystyrene sulfonate. Brijmohan et al.

[0151] Mass spectra were dominated by two novel singly charged ions of 183.0125 m'z and 199.0074 m / z in negative ionization mode (see Fig 1). These features are consistent with small NSPS fragments vinylbenzenesulfonate (C8H7O3S ) and hydroxylated vinylbenzenesulfonate (CsHyCUS'), respectively (see Fig 1). Few ions were detected in the range between 500 and 4000 m / z.

[0152] The ultraviolet (UV) absorption spectrum showed increasing absorption between 300 and 200 nm consistent with scattering and absorption features at -228 and -255 nm typical of polystyrene (see Fig 14) which shares similar carbon backbone structure to the basic repeating unit of Amberlite AIR 120 Na+and P-NaSS (Ps-100).

[0153] Dynamic light scatter (DLS) of the filtered solution of NSPS showed the formulation to be nanoparticulate with a hydrodynamic diameter of 159.09± 6.39 nm and a poly dispersity of 0.22 ± 0.02 (see Fig 15) consistent with an amphipathic polymer that consists of particles with a relatively narrow size distribution. Based on the DLS measurements, the present inventors estimate the size of NSPS to be at least 56 kilodaltons (kDa) and up to 770 kDa, assuming spherical structure .

[0154] In conclusion, NSPS formulation is a stable nanoparticulate that is soluble in aqueous media. It consists of associated monomers with a distribution of sizes. Regarding storage, NSPS should be refrigerated. Annual mass spectrometer tests of our formulation revealed that NSPS maintained its structural properties 1 year after formulation. No convincing evidence of masses 199 and 182 Da were found 2 years after formulation (see Fig 16).

[0155] To validate that the 199 Da monomer, which had not been known or found commercially, is the active ingredient in NSPS, the present inventors successfully synthesized a novel hydroxylated vinylbenzenesulfonate monomer of mass 199 Da (VU0945652) shown in Fig 2. Both the nanoparticulate and monomer were found to be efficacious in killing tumor cells containing extracellular HAP as described below.

[0156] NSPS increases pH in calcium phosphate phantoms. In the phantoms that contained 10 ml H2O and 1 mg calcium phosphate, pH increased in the presence of IX NSPS (~4 nmol / ml for 55 kDa molecular weight) within 5 min and this increase persisted over (1 week); pH at baseline of 10 ml H2O with or without 1 mg of CaPO4 salt was 6.23 ± 0.05 (mean ± STD) and 6.41 ± 0.11 after adding NSPS to the 10 ml H2O only (p>0.05) and significantly increased to 6.92 ± 0.11 (p<0.05) after adding NSPS to the H2O + CaPO4 mix.

[0157] NSPS dependent cytotoxicity is co-incident with cells lines containing microenvironmental HAP. Within 24 hours of adding the small concentration of IX of NSPS (~7 pM), significant tumor cell death (~ 10%, p<0.05) was observed in 4T1 and MDA-MB-231 cultures that contain TME-HAP. NSPS had minimal impact (minimal tumor cell death) on H292 cell cultures that lack significant detectable ECM-HAP (see Fig 17) while NSPS had no impact on the MCFlOa normal breast cells with cell death comparable to those receiving vehicle (p<0.05), see Fig 3. The present inventors attribute the lack of response to NSPS to the absenceof ECM-HAP in the MCFlOa cells. On the other hand, the small but significant effect of NSPS on H292 cells may suggest the presence of low levels of ECM-HAP in that cell line that is not revealed by our staining protocols. For the 4T1 cell line, the present inventors observed dose dependent cell death when testing varying (IX and 0.3X) concentrations of NSPS (see Fig 4). Greater efficacy on the tumor cells was obtained with ~16 pmol / ml of VU0945652 (see Fig 4). This high dosage of the small molecule VU0945652 is expected as at least 2 of the monomers would have to reach a HAP lattice simultaneously to chelate calcium as described above. However, the study demonstrates that VU0945652 is the active cytotoxic ingredient of NSPS polymer.

[0158] NSPS temporarily increases pHe in tumors with TME-HAP, in vivo. Using CEST MRI, the present inventors found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration, see Fig 5. As expected, no changes in pHe were detected in the muscle of the NSPS treated mice, in 4T1 tumors of mice receiving vehicle (controls), or in H292 tumors receiving NSPS due to the absence of TME-HAP in the H292 tumors (see Fig 18).

[0159] The pH in the ex vivo tumor homogenates of the NSPS-treated mice was 6.51 ± 0.04 (mean ± SD) and significantly higher (p<0.05) than the pH of the tumors from the vehicle treated mice (pH 6.19 ±0.1) or from untreated (sham) mice (pH = 6.20 ± 0.08) (see Fig 7). The pH of the homogenates is a measure of the average acidity that includes the extra and intracellular contents of the tumors and does not take into account tumor and extracellular heterogeneity, i.e., this study measured the macro pH of the tumor. Thus, the change in micro extracellular pH (i.e., the decreased acidity of the extracellular space) after NSPS treatment, is expected to be much larger than is observed in the tissue homogenates. Taken together, the in vivo pHe and ex vivo tumor pH results strongly suggest the acute alkalosis due to NSPS treatment.

[0160] In vivo efficacy of NSPS. The present inventors originally hypothesized that achieving tumor efficacy, in vivo, through dissolution of TME-HAP would be a two-step process: (i) create an injectable drug that could chelate one of the components of HAP, which inour case is NSPS to chelate calcium; and (ii) rapidly dissolve as much TME-HAP as possible to maintain a localized alkalosis status long enough to have a significant impact on the tumor without affecting normal cells. Based on the pHe results, a maximum practical dose of 2.4 mg NSPS dissolved in 0.2 ml saline was tested. This is the equivalent of nearly 100 mg / kg NSPS for an average mouse weight of about 25 g. To take advantage of the time it takes pHe to reset back to normal levels (~1 hour), the present inventors initially tested the following dosing regimen protocol: tumor bearing mice were treated with 25 mg / kg / 0.05 ml every ~1 hour up to a total of 100 mg / kg / 0.2ml. A quarter of the NSPS was injected intravenously (tail vein, jugular catheter, or retro-orbital) every hour 4 times till the entire 2.4 mg / 0.2ml were administered. This dosing regimen has proven to be highly efficacious in treating tumors containing TME-HAP with minimal adverse effects. Therefore, that dosing regimen was continued and is referred to this dosing regimen as one-time indicating that the 100 mg / kg of NSPS were administered in one day in under 6 hours.

[0161] In vivo NSPS dependent changes in mineralization were evaluated using18F-NaF. There was a significant reduction (p<0.05) in18F-NaF uptake post NSPS treatment as expected;18F- uptake in the tumor = 3.8 ± 0.5 %ID / g (percent of the injected dose per gram) at baseline compared to 1.8 ± 0.5 %ID / g following one-time treatment with 100 mg / kg NSPS (compare Figs 7A and 7B). These results are concordant with dissolution of at least 50% of TME-HAP by NSPS treatment. Note that because bone uptake of18F-NaF was -9 times higher than tumor HAP uptake due to the large skeletal surface area, the bone signal appears saturated in the18F-NaF PET images as discussed in previous work.18F-NaF uptake by HAP on skeletal bone surface appeared unaffected by NSPS treatment.18F-NaF uptake in skeletal bone was 30.2 ± 2.6 %ID / g pre-NSPS and 29.7 ± 2.4 %ID / g post NSPS (p>0.05). Of similar importance, is that18F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 hours of treatment with one-time NSPS (compare Figs 7C and 7D and see Table 1, below for quantitation) for the MMTV-Neu mice, which persisted for at least one week (see Fig 7E). These results indicate significantly reduced cellular glucose metabolic activity in the tumors after treatment with NSPS. No changes were found in the radiotracers’ uptake in the tumors of the control groups that received saline (see Figs 7F & 7G and Table 1). Harvested tumors following the 1- week18F-FDG scans revealed apoptosis (via cleaved caspase 3 staining, -75%) throughout the tumors of the NSPS treated mice with minimal proliferation (via Ki 67 staining, <25%), see Figs7H & 7J, respectively, indicating tumor cell death. No clear apoptosis signaling was found for the vehicle treated mice (see Fig 7K & 7L). On the other hand,18F-NaF uptake in the skeletal bone was 30.2 ± 2.6 %ID / g at baseline and 30.1 ± 2.4 %ID / g post NSPS (p>0.05) or vehicle (i.e. no noticeable changes in bone HAP).treatment with either 100 mg / kg / 0.2 ml of NSPS one-time or vehicle (0.2 ml saline one-time). Numbers displayed as mean ± SD. P values are for the differences in FDG uptake between baseline and post NSPS or vehicle administration in each organ.[00162J Similar results to those of the MMTV-Neu tumor mice in other cancer models including the MDA-MB-231 breast, 4T1 breast, PC3 prostate, and HCA-7 colon tumors were obtained (see Fig 19 and Table S2, below). All tumors were associated with TME-HAP as indicated using alizarin red S & von Kossa staining (see e g. Fig 19). Following one-time treatment with 100 mg / kg NSPS, the MDA-MB-231 were imaged 1 week later with FDG while the rest of the mouse models were imaged with FDG at 24 hours, then euthanized. IHC on the tumors revealed -10% apoptosis (cleaved caspase 3) and -90% Ki 67 at 24 hours post NSPS treatment (see Fig 19).Table S2. Broad applicability of NSPS on mouse models of cancer.18F-FDG uptake (%ID / g) in tumors of orthotopic mouse models at baseline and within 24 hours post onetime treatment with 100 mg / kg of NSPS. Values displayed as means (SEM).TIn each study, a control cohort consisting of the same number of animals was imaged at baseline and after vehicle (saline) injection, hi each study.18F- FDG uptake in the tumor was comparable to baseline values of the experimental cohort and uptake was unchanged following saline injection. ft Baseline18F-FDG in these tumors is lower compared to other tumor models due to the presence of central regions with low FDG uptake as shown in Fig. S7. ft This mouse model was used as negative control due to absence of detectable TME-HAP in the tumors.

[0163] While the H292 tumors were not detected with18F-NaF PET (see Fig 8A), they were naturally metabolically active and therefore, easily detected with18F-FDG PET (see Fig 8B). One-timelOO mg / kg NSPS had minimal effect on the subsequent uptake of18F-FDG (3.1 ± 0.4 %ID / g p>0.05, see Fig 8C). Additionally, IHC demonstrated no evidence of tumor cell death (see Figs 8D and 8E for cleaved caspase 3 and Ki 67 staining, respectively). These results are consistent with the absence of detectable TME-HAP in these tumors as revealed by the absence of significant von Kossa and alizarin red S staining in these tumors, see Figs 8F and 8G, respectively.

[0164] Tumor growth studies. Tumor growth was significantly slower (p<0.05) in the mice treated with one-time NSPS (see Fig 9). Using the standard exponential growth model in GraphPad Prism (v 5.0); Y = Y().ekxwhere Y is tumor size, X is time in days and k is growth rate constant, the tumor size doubling time was measured to be 7.1 and 5.7 days for the NSPS treated vs control mice, respectively. The treatment / control (T / C) tumor size ratios at day 21 = 39% ± 10% or ~ 40% reduction in tumor growth rate following one-time treatment with NSPS. Thereduction of tumor growth rate by NSPS is attributed to impacts on tumor metabolic activity and increased tumor cell death following treatment.

[0165] Biodistribution and toxicity studies.

[0166] Mass spectrometry of tissue samples. The following observations are noted: (i) NSPS monomers appear to be present without the hydroxyl group at a mass of 181.97 Da; (ii) Initial preliminary analysis detected NSPS signature in tumors, whole blood (but not plasma), bone and whole heart (perhaps attributable to whole blood content) at 10 min and 1 hr (see Fig 20). However, NSPS was detected in the tumors only at 4 hours but not in bone or whole heart. No significant NSPS signature was detected in the liver, spleen, muscle, kidneys, and brain extracts at any time. This lack of detection may be an issue of sensitivity of some of the tissue extracts. As expected, the present inventors found no NSPS signature for tumors from mice that received vehicle (see Fig 20(F).

[0167] Bone toxicity studies. No significant differences (p>0.05) in any of the bone microCT parameters measured across the midshaft (see table 2, below and Fig 10) or in the trabecular bone (see table 3 and Fig 10) were found. Thus, NSPS, even at 100 mg / kg, had minimal impact on bone mineralization and bone growth.Table 2: NSPS has minimal impact on bone mineralization. Toxicity measurements on femur bone harvested from female white balb / c mice 10 days following injection of one-time 100 mg / kg NSPS or vehicle (saline). Data presented as mean ± SD (n = 10 per group), p > 0.05 between the two groups for any measurement.

[0168] Plasma calcium and phospates levels. There were no signifcant differences in calcium or phophates levels of the NSPS treated versus the control mice at 24 hours; Ca2++levels in plasma of NSPS treated and control mice were 0.43 ± 0.01 and 0.42 ± 0.02 pg (mean ± SE) (p = 0.620), respectively. Plasma phosphates levels were 2.89 ± 0.09 and 2.78 ± 0.28 mmol / L (p = 0.347), in the NSPS treated and controls, respectively. Cation exchange is a rapid and reversible process where cations like calcium are exchanged on an equivalent charge basis, i.e. Na++for Ca++and vice versa. NSPS has the potential to temporarily alter local calcium equilibria but does not cause systemic calcium deficiency.

[0169] Nephrotoxicity. No signs of fibrosis or renal dysfunction were observed in the "mTc-MAG3 SPECT studies (see Fig 11-A). Additionally, contrast CT showed normal renal cortex and renal pelvis (see Fig 11-B). Thus notably, there was no evident nephrotoxicity due to NSPS treatment, results consistent with the absence of evidence of HAP in the renal microenvironment.

[0170] Example 3

[0171] This example shows additional calculations for an example of a NSPS polymer of the present invention.

[0172] (a) Approximate Number of Repeating Units. The molecular weight of an example of a polymer of the invention is about 55 kDa (55,000 g / mol).

[0173] Assuming one CH linker per monomer unit, the effective molecular weight per repeating unit is:Effective Weight 184 g / mol (monomer) 4 13 / mol (linker) 197 g / molPolymer Molecular Weight > 55, 000Number ol U nits — . ' . . « 279 unitsEffective Weight .197" '

[0174] (b) Number of linkers. In the example where there is one (CH)nlinker per unit, the number of CH linkers in the polymer is approximately: (CH Linkers = Number of Units- 1 « 279- 1) = 278

[0175] A 55 kDa NSPS polymer of VU0945652 will have approximately 278 CH linkers

[0176] The degree of polymerization is the number of repeating monomer units in the NSPS polymer ~ 279

[0177] (c) Charge Density. The polymer’s sulfonate groups (-SO3 ) contribute negative charges, which bind cations like calcium (Ca2+). Calculating the charge density using Sulfonate Groups ~ 279 and Polymer Weight = 55,000 g / mol

[0178] Charge density = 279 / 55,000 = 0.005 mol / g

[0179] (d) Surface Charge Density of NSPS. 1. Charge Contribution (Each sulfonate group (-SO3 ) contributes -1 charge; Number of sulfonate groups: 279 (for a 55 kDa polymer). 2. Polymer Size (The molecular weight (55 kDa) gives us the approximate mass per polymer molecule: 55,000 g / mol; the hydrodynamic radius (RhR hRh) or molecular dimensions from Dynamic Light Scattering (DLS) or modeling can help us estimate the surface area.) 3. Charge per Molecule: (total charge on one polymer molecule: Q = -279 e, where e is the elementary charge (1.602x 10-19 C). 4. Estimate Surface Area (assuming NSPS is spherical (an assumption), the surface area can be approximated as:, where Rh is the hydrodynamic radius = 160 nm (from DLS); A = 3.22 x 10‘13m2. 5. Total Charge on the Polymer: The total negative charge on the polymer is due to the 279 sulfonate groups (-SO3 ): Q=-279 e; Substitute e=l.602*10’19C; Q=-279 x 1.602 x 1019=-4.47* 1017. 6. Surface 2Charge Density: The surface charge density (c) is given by:where o = -1.39 X 10-4C / m2.

[0180] (e) Calcium Binding Capacity: If the sulfonate groups bind calcium ions(Ca2+\text{Ca}A{2+}Ca2+), calculate the maximum binding capacity: each sulfonate group can bind one Ca2ion; Total binding capacity:Ca* ' Binding Sites ™ Number of Sulfonate Groups ™ 279

[0181] Binding in moles: Binding capacity = 279 / 6.022 x 1023 = 4.63 X 10-22 mol / NSPS polymer molecule

[0182] (f) Benchmarks for Comparison. Ion-exchange resins (like Kayexalate or sodium polystyrene sulfonate) typically bind ~l-2 moles of cations per mole of polymer (depending on crosslinking and surface area). Kayexalate has a comparable charge density since it is also sulfonate-based, binding potassium (K+) ions in the gut.

[0183] An example of the NSPS polymer, with 0.005 mol / g, has a reasonable binding capacity for calcium ions, especially for applications where the binding of divalent ions (e.g., Ca2+) is desired. The binding capacity is comparable to other effective ion-exchange materials.

[0184] (g) Calculation of NSPS size: To estimate the size of particles in a sample with a reported hydrodynamic diameter (d) of 159 nm and a polydispersity index (PDI) of 0.224, assuming a spherical shape and a density of 1 g / cm3, we can use the Stokes-Einstein equation: d = k * (k b * T) / (6 * pi * eta * r_h) where d is the hydrodynamic diameter, k is the Boltzmann constant, k b is the temperature, eta is the viscosity of the medium, and r_h is the hydrodynamic radius of the spherical particle.

[0185] Assuming room temperature (T = 298 K), the Boltzmann constant (k) is 1.38 x 10A- 23 J / K, and the viscosity of water at room temperature is about 0.89 cP or 0.89 x 10A-3 Pa s.

[0186] Rearranging the equation to solve for the hydrodynamic radius (r_h), see: r_h = k * (k_b * T) / (6 * pi * eta * d)

[0187] Substituting the given values, see: r_h = (1.38 x 10A-23 J / K) * (298 K) / (6 * pi * (0.89 x 10A-3 Pa s) * (159 nm)) r_h = 7.74 x 10A-9 m or 7.74 nm

[0188] This calculation gives an estimated hydrodynamic radius of about 7.74 nm for the spherical particles in the sample. However, it's important to note that this calculation assumesthat the particles are perfectly spherical and have a density of 1 g / cm3, which may not be the case in practice. Additionally, other factors such as particle shape, surface charge, and interactions with the solvent can affect the hydrodynamic diameter measured by DLS, so additional characterization techniques may be necessary to confirm the size and properties of the particles.

[0189] Example 4

[0190] This example shows the inventive (NSPS) in treating ovarian cancer via breakup of tumor associated hydroxyapatite (HAP).

[0191] Ovarian Cancer is Associated with Poor Outcomes. The lack of effective screening strategies often leads to late-stage diagnosis, which combined with the toxicity of current chemotherapeutics, limits the frequency and dosage of treatment. Additionally, many patients develop resistance to therapies, including surgery and novel therapeutics like PARP inhibitors, resulting in a high relapse rate and poor long-term survival. Patient outcomes are also influenced by disease extent and overall health, which can affect the feasibility of primary surgery. Complicating treatment choices further, conventional imaging tools may lack clear criteria for selecting between initial surgery and neoadjuvant chemotherapy. Despite advances in targeted therapies and immune-modulating therapies, success rates remain limited due to the heterogeneous nature of tumors and the immunosuppressive ovarian cancer tumor microenvironment (TME).

[0192] TME acidosis contributes to resistance. A key factor contributing to resistance to treatments is the acidosis conditions in the TME. This is mostly due to metabolic reprogramming in tumors which results in overproduction of H+ions (and lactate), promoting tumor survival and progression. While neutralizing the acidity of the TME has been proposed to improve anti-tumor response to therapy and potentially limit the ability of tumor cells to develop drug resistance, previous methods such as systemic bicarbonate treatment or inhibition of specific receptors have resulted in confounding systemic effects, including changes in whole body pH and metabolism, extreme fatigue, seizures, or irreversible normal tissue damage. There is a pressing need for developing new therapeutics and methodologies to identify subsets of tumors predicted to respond to particular treatments.

[0193] TME-HAP; a target for ovarian cancer treatment. The injectable bridged cation exchange nanoparticulate sulfonated polystyrene solution (NSPS), ~55 kilo Daltons, for cancer treatment of the present invention, is shown to target and breakup TME-HAP reversing TME- acidosis without impacting whole body pH. The primary component of NSPS is the monomer VU0945652, a vinylbenzenesulfonate (CsHvC S’ [M-H]) with mass = 199.0074; NSPS (soluble in saline or PBS) chelates calcium, via wrap-around method, from the TME-HAP lattice and in return release (PO^-) and OH' anions; 2NSPS++ Ca++NSPS2Ca + 2Na+. The abrupt increase in and OH' in the TME and / or the potential formation of NaOH results in an acute alkalosis localized to the TME eventually leading to inhibition of tumor cell metabolism, tumor growth, and tumor cell death with minimal adverse effects as HAP is absent in normal soft tissue. Cation exchange is a rapid and reversible process exchanging cations (e.g. Ca++) on an equivalent charge basis, i.e. Na++for Ca++and vice versa. While NSPS may potentially temporarily alter local calcium equilibria, the present inventors found no evidence of NSPS- induced systemic calcium deficiency or elevated phosphate levels. The released calcium and phosphate levels following break up of TME-HAP may be too modest to cause detectable changes in blood concentrations of these molecules. Moreover, these ions in the TME could be reabsorbed by tumor cells through transcellular and paracellular pathways.

[0194] A switch from acidosis to alkalosis can be toxic to tumors and an area of great interest in cancer therapy. However, perturbation of only tumor extracellular pH and not systemically (whole body) is challenging and, to our knowledge, has not previously been achieved. The strategy of the present invention is distinct from other methods in that it is designed to target and breakup HAP in the TME to temporarily elevate tumor extracellular pH alone without changing whole body pH, attributed to the absence of HAP in normal soft tissue. This approach can provide highly selective therapy of aggressive HAP -producing ovarian tumors. There are no other known injectable compounds that can break up TME-HAP in vivo, making NSPS a one-of-a-kind and first in a class of novel cancer therapeutics.

[0195] Using mass spectroscopy, the present inventors detected NSPS in tumors and whole blood within 1 hours post i.v. administration in orthotopic mouse models of cancer that is cleared within 4 hours via the gallbladder. This is one of the advantages of macromolecular (>1 kDa) drug formulations like NSPS which are likely to spare healthy tissue from toxicity bypreferentially accumulating within the TME through the enhanced permeation and retention (EPR) effect that appears mediated by disorganized tumor vasculature and poor lymphatic drainage of tumors. The present inventors attribute the limited interaction of NSPS with bone to a combination of factors including the compact structure of HAP on bone surface, large skeletal surface area, absence of EPR and large vasculature to bone surface, and continuous bone remodeling. By contrast, the diffused and scattered nature of TME-HAP, combined with the large and disorganized tumor vasculature, afford susceptibility to calcium chelation and dissolution by NSPS.

[0196] When NSPS, was developed, a quantity of 2.4 mg (-100 mg / kg for an average mouse weight of 25 g). This amount, dissolved in 0.1 ml of saline or PBS, was consistently used across all subsequent in vivo studies (69), including the preliminary data below on mouse models of ovarian cancer.

[0197] Generally, polymer-based drugs are administered at high doses (up to thousands of mg / kg) and / or as a flat dose instead of mg / kg to achieve therapeutic efficacy due to the intrinsic physicochemical properties that may limit circulation time in the blood plasma and / or bioavailability. Therefore, the use of 2.4 mg of NSPS is not only reasonable but is considered a low dose within the nanoparticle dosing spectrum. Polymeric macromolecular drug formulations (like NSPS) are likely to spare healthy tissue from toxicity by preferentially accumulating within tumors, through the enhanced permeation and retention (EPR) effect that appears mediated by disorganized tumor vasculature and poor lymphatic drainage in tumors

[0198] Clinical significance of TME-HAP and ovarian cancer: High-grade serous cancer (HGSC) accounts for over 70% of ovarian cancer deaths and more than 80% of advanced-stage ovarian cancer diagnoses worldwide. Examples of HGSC biopsies from the TMA’s that are positive for TME-HAP (TME-HAPpos) and negative for TME-HAP (TME-HAPneg) are shown in Fig. 21 at 40X magnification. At this level of magnification, clusters or lattices of HAP molecules within the TME appear as dark spots, with sizes ranging from a few microns to a few millimeters. As demonstrated in previous breast cancer studies, these HAP clusters can eventually be detected as calcium deposits via CT as they grow in size. However, individual HAP molecules are often diffused throughout the TME. Thus, tumors classified as TME-HAPnegmay actually contain scattered HAP not visible microscopically. Moreover, given the variability in tumor and matrix distribution and the limitations of biopsy sampling, the actual prevalence of TME-HAP might be underreported in our patient group.

[0199] Since18F‘ ions bind to HAP molecules (~9.4 angstroms), using18F-NaF PET imaging can detect scattered TME-HAP before it becomes visible on CT scans, identifying ovarian cancer patients who may respond to NSPS treatment. Additionally, CT cannot differentiate between types of calcium deposits, such as calcium oxalate and hydroxyapatite (HAP), whereas18F-NaF specifically binds to HAP but not oxalate.

[0200] TME-HAP was more prevalent in serous cancer, both HGSC and low-grade serous cancer (LGSC) than other types of ovarian cancer. This is consistent with recent clinical findings by Wen et al..

[0201] Patients with TME-HAPpostumors had poorer outcomes, e.g., shorter overall survival (OS) and disease-free interval, compared to patients with TME-HAPnegtumors regardless of their response (sensitivity) to platinum chemotherapy (see Fig. 22 and see Table 02 in Outcomes). These results suggest that TME-HAP may serve as a potential biomarker for predicting platinum chemotherapy response. Notably, HGSC patients with TME-HAPpostumors may be less responsive than patients with TME-HAPnegtumors, a clinically significant discovery.

[0202] Almost 35% of the serous cancer patient population (i.e. 1 out of every 3 patients) presented microscopically detectable TME-HAP. This sizable subgroup could potentially be at higher risk (see Fig. 22) with potential to benefit from HAP -targeted therapy.

[0203] The NSPS of the present invention proved to be a promising novel drug demonstrating anti -turn or efficacy and rapid TME-HAP dissolution in ovarian cancer mouse models. For this example, the animal model of ovarian cancer to test NSPS efficacy was mouse ID8-luciferase tagged tumor cells (5 xlO6) injected intraperitonially (i.p.) in immune competent female C57BL / 6 mice. This is an established syngeneic model of ovarian cancer which recapitulates both the extensive peritoneal dissemination and ascites development seen in humandisease within 4 weeks. This model allows assessment of tumor burden using bioluminescence imaging (BLI).

[0204] Assessing NSPS efficacy in vivo using BLI. ID8-luc cells (5 xlO6) were injected intraperitoneally (i.p.) into immunocompetent 8-week-old female C57BL / 6 mice (n = 15) divided into three cages of five mice each. After four weeks, the inventors performed bioluminescence imaging (BLI) at 10 minutes after intraperitoneal injection of 150 mg / kg D- luciferin (Perkin Elmer), which the present inventors determined to be the ideal imaging window through kinetic analysis calibration curves in this animal model. Each cage of five mice received luciferin simultaneously and was imaged simultaneously using our IVIS scanner (Perkin Elmer). The next day, the mice were randomly divided into two groups, with each group receiving either a one-time intravenous (i.v.) injection of 2.4 mg of NSPS (n = 8), equivalent to -100 mg / kg for an average mouse weighing 25 g, or vehicle (PBS; n = 7). Each cage contained two to three NSPS and two to three PBS injected mice. This randomization ensured robustness and avoided any bias in post-treatment imaging. The mice were imaged again weekly at weeks 5, 6, and 7 post tumor cell injection to assess tumor growth and progression in response to NSPS.

[0205] A 15% reduction in luminescence in the NSPS-treated mice was observed compared to an 11% increase in the control group at week 5 (p = 0.0302), see Fig. 23. While both groups showed increased luminescence over time, the vehicle-treated controls had a 5-fold increase from week 5 to week 6, whereas the NSPS group showed only a slight increase, indicating a significantly reduced tumor growth rate. These results suggest that a one-time NSPS treatment slows tumor progression and metastasis, aligning with our previous findings of a 40% reduction in orthotopic tumor growth in mouse models of breast cancer models.

[0206] Assessing NSPS efficacy in vivo using18F-NaF PET imaging. Four weeks after ID8-luc tumor cells were injected into female C57BL / 6 mice (8 weeks old), tumor burden was confirmed through BLI imaging. Subsequently, mice were administered an i.v. injection of ~17 MBq of18F-NaF, followed by a PET imaging session 60 minutes later, which lasted for 20 minutes, followed by a CT scan at a nominal resolution of 0.14 mm. The next day, the mice were randomly divided into two groups; one group received an intravenous injection of 2.4 mg of NSPS (n = 8), and the other group was treated with vehicle (PBS) as controls (n = 8). Within 24hours of treatment, the mice underwent18F-NaF PET imaging following the same protocol as baseline. The PET images were normalized to the injected dose and decay corrected to the time of injection. They were also corrected for attenuation and scatter. To quantify the radiotracer uptake, the present inventors drew three-dimensional regions-of-interest (ROIs) around the peritoneal tumors in the PET images and determined the mean radiotracer activity in units of percent injected dose per gram (%ID / g) for each mouse. After two weeks, the mice were euthanized, and the tumors along the peritoneal wall were harvested for immunohistochemistry (IHC) analysis. Ki 67 staining was used to evaluate tumor cell proliferation, while cleaved caspase 3 was used to assess apoptosis. Additionally, von Kossa and alizarin red S were used to evaluate hydroxyapatite (HAP) deposition. The following results were obtained:

[0207] 1. Display Settings: Consistent with our previous work, skeletal bone uptake of18F-NaF was approximately nine times greater (36 ± 4 %ID / g) than TME-HAP uptake. This is attributed to the large bone surface area and the ability of18F‘ ions (1.33 Angstroms) to reach HAP on the bone surface through the bone marrow. To visualize the tumors, the maximum intensity projection window was set to 3 %ID / g resulting in the bone signal to appear saturated in the displayed PET images (see Fig. 24 panels A and B).

[0208] 2. Detection of Tumor Microcalcification: Tumor microcalcification, resulting fromTME-HAP progression, was not yet detectable via CT at the resolution (0.14 mm) used (see panel C in Fig. 24), but was easily detected

[0209] with18F-NaF PET imaging. Although PET has a spatial resolution > 1 mm, the clinical benefits of using18F-NaF PET imaging for detecting peritoneal tumors via TME-HAP outweighs this limitation.

[0210] 3. Rapid Disruption of Tumor Microenvironment: The present inventors found a-50% reduction in18F-NaF following treatment with NSPS compared to baseline (compare panels A and B in Fig. 24) where18F-NaF uptake in the peritoneal cavity was 4.29 ± 0.89 %ID / g (mean ± SEM) and 2.33 ± 0.66 %ID / g at baseline and post NSPS treatment, respectively (p = 0.022). On the other hand, there was a slight increase in18F-NaF uptake in the control tumors; 4.33 ±0.90 %ID / g and 4.49±0.51 %ID / g at baseline and post PBS injection, respectively. These results indicate that NSPS effectively disrupts the peritoneal TME, specifically targeting andbreaking down tumor-associated HAP. The 24-hour response to treatment suggests that NSPS has a rapid impact on TME-HAP. These results are also in concordant with our previous work on orthotopic mouse models of breast cancer.

[0211] 4. TME-HAP is associated with ID8-luc tumors: The ID8-luc tumors were positive for von Kossa and alizarin red S confirming that they are TME-HAPpos(see Fig. 24 panel F).

[0212] 5. NSPS inhibits tumor cell proliferation. Ki-67 was observed only along the circumference of the tumors treated with NSPS, whereas in the control group (PBS-injected), Ki- 67 was found throughout the tumors (see compare panels G and H in Fig. 24 and see Table 4).Table 4. NSPS induces tumor cell apoptosis and while limiting tumor cell proliferation. Staining of ID8-luc peritoneal tumors harvested from mice treated with 100 mg / kg NSPS or vehicle (saline).

[0213] 6. NSPS induces tumor cell apoptosis', cleaved caspase 3 was significantly higher in the NSPS-treated tumors but minimal in the controls, see Fig. 25 panel J and Table 4.

[0214] 7 NSPS treated mice had smaller tumors'. While statistical significance wasn't reached, attributed to the small sample size, the peritoneal tumors in the NSPS-treated mice were 45 ± 15 % smaller compared to the controls (see Table 4 and panel H of Fig. 24).

[0215] Onetime treatment with NSPS mimics weekly cisplatin platinum chemotherapy treatment. Four weeks post i.p. injection of ID8-luc tumor cells in female C57BL / 6 mice (8 weeks old, n = 20), the present inventors imaged the mice at baseline with 18F-NaF PET as described above. The following day, the mice were randomly divided into 4 groups (n = 5 per group) and received one of the following treatments: (a) Group 1 received weekly i.v. injectionsof PBS (controls) for four weeks (weeks 4, 5, 6, and 7 post tumor cell injection); (b) Group 2 received weekly i.p. injections of 2 mg / kg cisplatin chemotherapy (weeks 4-7); (c) Group 3 received 2 mg / kg cisplatin weekly for 2 weeks (weeks 4 and 5), then switched to weekly PBS injections (weeks 6 and 7) as a model of tumor recurrence; and (d) Group 4 received a one-time i.v. injection of NSPS (2.4 mg). All mice were then imaged weekly using the same 18F-NaF PET imaging protocol up to 8 weeks post tumor cell injection. ROIs were drawn around the peritoneal tumors, and time-activity curves (TACs) of 18F-NaF binding to TME-HAP were established for all mice. Results of this study include:

[0216] 1. Group 1 (PBS; controls): had a continuous upward trend in their TACs attributed to increased tumor growth and metastasis within the peritoneal cavity (see Fig. 25).

[0217] 2. Group 2 (weekly 2 mg / kg cisplatin): had a downward trend in their TACs suggesting decreased available TME-HAP attributed to inhibition of tumor growth and / or tumor regression.

[0218] 3. Group 3 (2 weeks of cisplatin + 2 weeks of PBS): their TACs initially had a downward trend following cisplatin treatment, followed by a one-week quasi flat curve and then an upward trend in the TAC after week 6 when switching to PBS attributed to tumor recurrence and / or discontinuation of tumor inhibition.

[0219] 4. Group 4 (only onetime NSPS): the present inventors detected a downward trend in the TAC similar to that of weekly 2 mg / kg cisplatin treatment and this trend persisted for at least 3 weeks before going up again by week 8 further supporting the long-term tumor growth inhibitory effects of NSPS with minimal adverse effects.

[0220] This preliminary study validated18F-NaF PET imaging as a non-invasive imaging tool to track the treatment response of TME-HAPposovarian tumors. This has significant clinical implications, as it provides a metric of tumor burden regardless of the type of treatment.

[0221] Given the toxicity and adverse effects associated with platinum chemotherapy, the present invention shows that NSPS will synergizes with current standard of care treatment, such as cisplatin. This synergy could allow for significantly reduced chemotherapy dosing and / orfrequency, thereby minimizing toxicity and adverse effects typically associated with cisplatin and other standard-of-care therapeutics in the clinic.

[0222] NSPS induce ovarian tumor cell death in vitro. ID8-luc cells were cultured in DMEM media with or without an osteogenic cocktail that consisted of 50 pgmE1ascorbic acid and 10 pM beta-glycerophosphate (UG) for at least 21 days similar to the work of others. Aliquots of the cell cultures were then stained with alizarin red S and von Kossa which revealed the presence of detectable HAP in the in vitro extracellular matrix (ECM-HAP), when cultured with the osteogenic cocktail media (OCM), see Fig. 26, but not the ones cultured without the OCM (not shown). Then the ID8-luc cells were seeded in 96 well plates. At -70% confluency, NSPS ranging between 0.01 to 100 pM was added to the cells (in triplicate) or vehicle (PBS). For negative controls, the present inventors used MCFlOa normal breast cells that lack extracellular HAP regardless of the contents of the culture media. Within 24 hours after applying NSPS or vehicle, the present inventors measured the number of total cells and dead cells using a hemocytometer and trypan blue. The present inventors observed 1% (p>0.05 compared to controls), 50 % (p>0.05), and 99% (p>0.05) ID8-luc tumor cell death cultured with the osteogenic cocktail media (OCM) at -0.01 pM, 43 pM, and 89 pM of NSPS, respectively. Regarding controls, MCFlOa normal breast cell death was <1% at any NSPS dose, while ID8-luc tumor cell death cultured in normal DMEM without OCM was 4% at NSPS concentration of 89 pM (p>0.05 compared to MCFlOa at 89 pM of NSPS) and 1% death at NSPS concentration of 10 pM. The absence of cell death of the normal breast MCFlOa cells at any NSPS dose, demonstrate the safety of NSPS to normal cells. The slight tumor cell death of the ID8-luc cells cultured without OCM may suggest some production of ECM-HAP by 21 days. The tumor cell death of ID8-luc cells cultured with OCM within 24 hours of adding NSPS demonstrate the rapid efficacy of NSPS. By contrast, polymer-based drugs typically take days to achieve as much as 10% efficacy even at high concentrations. For example, PAPBN, an oxidation sensitive copolymer, required 1 mg / ml to achieve <1% efficacy on mouse breast 4T1 cells in culture within 24 hrs, and about 10% efficacy after 72 hours. Only when combined with Doxorubicin chemotherapeutic and a 48 hr incubation, was a 90% efficacy achieved by both drugs.

[0223] Taken altogether, the results present strong evidence of the efficacy of NSPS in the treatment of ovarian cancer. The advantages of our approach include: (i) Ubiquity: any type ofovarian cancer malignancy with TME-HAP, is expected to respond well to treatment with NSPS regardless of the tumor type or stage; (ii) Response time: response to treatment can be observed within 24 hrs after administration of NSPS, (iii) Lack of systemic toxicity: the present inventors found no evident NSPS toxicity in normal tissue or bone; (iv) Treatment tracking: HAP -binding radioligands can be used to track dissolution of TME-HAP with NSPS.

[0224] There are FDA-approved cation exchange sulfonated polystyrene drugs such as Kayexalate, used for hyperkalemia treatment at dosages up to 800 mg / kg. However, Kayexalate, linear polymer, lacked therapeutic effects on our tumor models due to negligible affinity for calcium.

[0225] Establishing platinum chemoresistant ovarian cancer models. The present inventors successfully generated a cisplatin-resistant clone of ID8-luc cells (ID8-luc CP) by chronic treatment of parent cells (ID8-luc PAR) with cisplatin (10 pM) (see Fig. 27). The present inventors have validated robustly reduced cisplatin response compared to the parental cells in growth culture assays (see Fig. 27A) and on indices of tumor burden including BLI (see Fig. 27B-C), harvested tumor weight (see Fig. 27D), and volume of ascites (see Fig. 27D) in immunocompetent female C57B1 / 6 mice.

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[0227] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

We claim:

1. A cation exchange nanoparticulate sulfonated polystyrene solution comprising a polymer, the polymer having at least one benzenesulfonate monomer and at least one Ca2+ calcium ion.

2. The solution of claim 1, wherein the monomer is of the following formula:wherein n is an integer with a total value of from about 50 to about 300.

3. The solution of claim 1, wherein the monomer is of the following formula:wherein x is 0-6, and >'vvv' is an attachment point for another monomer.

4. The solution of claim 1, wherein the mass of the monomer is about 199 Da ± 20 Da.

5. The solution of claim 1, wherein the molecular weight of the polymer is about 45-65 kDa, and up to about 800 kDa.

6. The solution of claim 1, wherein the number of monomer units is about 200-300.

7. The solution of claim 1, wherein the polymer has a calcium binding capacity of about 4.63 X 1-22 mol per polymer molecule.

8. The solution of claim 1, wherein average hydrodynamic diameter of the of the nanoparticulate is about 160nm ± about 20 nm, preferably about 160 nm ± about 6 nm.

9. The solution of claim 1, wherein the poly dispersity is about 0. 1 - 0.5, preferably about 0.22 ± 0.02.

10. The solution of claim 1, wherein the polymer is of the following formula:wherein < vw' is an optional attachment point for another monomer.

11. The solution of claim 1, further comprising saline and EtOH.

12. A method for treating cancer in a subject, comprising: administering a therapeutically effective amount of a solution of claim 1.

13. The method of claim 12, wherein the administration step is injectable.

14. The method of claim 12, wherein the cancer is a tumor that has a microenvironment that comprises hydroxyapatite.

15. The method of claim 14, wherein the cancer is breast cancer, prostate cancer, colon cancer, or ovarian cancer.

16. The method of claim 12, wherein the solution of claim 1 is co-administered in combination with one or more optional therapeutic agents.

17. The method of claim 16, wherein the one or more optional therapeutic agents includes an anti cancer agent.

18. The method of claim 17, wherein the anticancer agent is cisplatin.

19. A pharmaceutical composition comprising the solution or a polymer of claim 1, and a pharmaceutically acceptable carrier.

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