USE OF ANTIOXID ANT-LOADED NANOZYMES TO TREAT CANCER

RELATED APPLICATION

This application claims priority to United States Provisional Application No. 62/321 ,040 that was filed on April 1 1 , 2016. The entire content of the application referenced above is hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under 1 P50CA174521 -01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

One of the most promising new therapies for treating cancer involves the use of molecules (e.g., peptides) that are designed to bind selectively to cancer cells but not to noncancerous cells. To kill cancer cells selectively, a radioactive atom can be attached to the molecule, so that when the molecule binds to the cancer cell in the body, the radiation emitted kills the cancer cell. Because the molecules are designed to selectively bind to cancer cells, they selectively deliver the radiation dose to the cancer cells and not to other cells. One caveat that limits the approach is that because the kidneys are a filtration system that filters out a variety of molecules for a variety of reasons, the molecules for this type of targeted radiation therapy often accumulate in the kidneys and are filtered out. This causes unwanted radiation damage in the kidneys, which are critical organs. Accordingly, improved methods are needed.

SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION

Provided herein are nanoparticles loaded with antioxidants that are designed to (1) accumulate in the kidneys and bladder (2) localize antioxidant enzymes to the kidneys that will reduce radiation damage and (3) block accumulation of the radio-labeled molecules in the kidneys.

The inventors have surprisingly demonstrated that the formulations described herein containing nanozymes accumulate almost exclusively in the kidneys; significantly reduce kidney uptake of a radiolabeled peptide; and significantly increase the accumulation of the radiolabeled peptide in the tumor. While many of the results provided herein relate to the delivery and accumulation of antioxidants in the kidneys, the results provided herein indicate that other molecules, such as proteins or antibodies, can be similarly and specifically targeted to the kidneys. Certain embodiments of the present invention provide the use of a nanozyme that comprises an antioxidant in combination with a radionuclide based agent to treat cancer.

Certain embodiments of the present invention provide the use of a nanozyme that comprises an antioxidant to prevent or decrease renal toxicity.

Certain embodiments of the present invention provide the use of a nanozyme for administering a therapeutic agent to the kidney.

Certain embodiments of the present invention provide a composition that comprises a nanozyme comprising an antioxidant, a radionuclide based agent for treating cancer, and at least one pharmaceutically acceptable carrier.

In certain embodiments, the nanozyme is a PEI-based nanozyme, a PLL-based nanozyme, or a DET-based nanozyme.

In certain embodiments, the nanozyme is a PEI-based nanozyme.

In certain embodiments, the nanozyme is a PLL-based nanozyme.

In certain embodiments, the nanozyme is a DET-based nanozyme.

In certain embodiments, the use or composition comprises a combination of a PEI-based nanozyme, and/or a PLL-based nanozyme, and/or a DET-based nanozyme.

In certain embodiments, the nanozyme comprises an amino acid and/or amino acid-like residue that includes a sulfhydryl functional group.

In certain embodiments, the antioxidant is a superoxide dismutase.

In certain embodiments, the antioxidant is a catalase or other antioxidant enzyme.

In certain embodiments, the use or composition further comprises the use of an endoplasmic reticulum (ER) stress-relieving agent.

In certain embodiments, the ER stress-relieving agent is 4-phenylbutyric acid (PBA).

In certain embodiments, the use or composition further comprises the use of amifostine.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Figure 1 depicts the use of nanozymes as radioprotectors for peptide-targeted radionuclide therapy (e.g., peptide-targeted radionuclide therapy). The kidney is the major excretion route as well as the dose-limited organ for peptide-targeted radionuclide therapy. The nanozymes described herein that include at least one antioxidant can protect the kidneys by scavenging the ROS generated by radio-peptide and fragments in the kidneys. Figure 2. Figure 2 depicts an example of a nanozyme useful in the practice of embodiments of the invention.

Figure 3. Nanozymes loaded with active antioxidant enzymes accumulate specifically with high efficiency in the kidneys of mice following tail vein injection. The first bar of each set of bars represents 0.5 h, the second 1 h, and the third 2 h.

Figure 4. Nanozymes loaded with active antioxidant enzymes accumulate specifically with high efficiency in the kidneys and are excreted through the bladder of mice and do not accumulate in tumors induced subcutaneous ly following tail vein injection. The first bar of each set of bars represents 0.5 h, the second 1 h, and the third 2 h.

Figures 5A and 5B. Nanozyme coinjection improves the biodistribution of radiopeptide

[ ²⁰³ Pb]DOTA-VMT-MCRl for imaging and therapy for melanoma.

DETAILED DESCRIPTION

Molecular constructs have been designed that encapsulate active antioxidant enzymes, such as superoxide dismutase and catalase. These constructs can be designed to accumulate in specific organs to deliver the active antioxidant enzymes to reduce oxidative stress related toxicities and damage to tissues in the organ tissue in which they have accumulated. In the field of targeted radionuclide based therapy for cancer, this is important because the kidneys are a dose limiting organ. Preliminary in vivo data in mice show that nanozymes loaded with superoxide dismutase accumulate selectively in the kidneys and bladder and do not accumulate in tumors of cancerous tumor-bearing mice. Surprisingly, the co-injection of nanozymes with a radiolabeled peptide resulted in decreased kidney retention of the radiolabeled peptide and increased tumor accumulation of the radiolabeled peptide. This indicates that the nanozymes can be used to increase the total radiation dose that can be administered for peptide and other molecule targeted radionuclide based therapies.

Accordingly, provided herein are nanoparticles that include polyethylene glycol and amino acid sequences that can be loaded with antioxidant enzymes, such as catalase and superoxide dismutase. Results show that these nanozymes can be designed to selectively accumulate in the kidneys so as to localize active antioxidant enzymes in the kidneys. The presence of localized antioxidants is expected to dramatically reduce renal toxicity associated with unwanted accumulation of radiolabeled peptides in the kidneys in targeted radionuclide based therapies for cancer. It is further expected that these nanozymes will also block peptides from accumulating in the kidneys. Importantly, data also show that the nanozymes do not accumulate in tumor xenografts in mice. Thus, these nanozymes can greatly reduce the renal toxicity associated with targeted radionuclide therapy, potentially enabling injection of doses a factor of 2 higher or more (e.g. , 10, 20, 50 or 100) than can be administered without the nanozymes because the kidneys are a dose limiting organ for this type of therapy. Applicant is not aware of any current technology that combines blocking of peptide uptake in the kidneys with delivery of antioxidants. Further, currently-available technologies provide only incremental improvement over radionuclide therapy in the absence of their use. In addition, administration of the current technologies for this application causes nausea and vomiting and immune responses that are potentially severe. Preliminary experiments with these nanozymes showed no signs of toxic effects, presumably because a bolus injection of blocking agent is not required because the nanozymes degrade slowly with an in vivo half life of approximately 45 minutes.

In one aspect, administration of the nanozymes encapsulating superoxide dismutase or other antioxidant enzyme(s) may be combined with administration of amifostine to facilitate kidney protection or protection of cells in the kidneys.

In one aspect, a poly-amino-PEG wrapper of the nanozyme that encapsulates the active enzyme component(s) of the structure includes amino acid and/or amino acid-like residues that include sulfhydryl functional groups that are slowly released as the 'wrapper' is degraded by biological processes, thereby slowly delivering antioxidants with the active enzymes. Amino acid-like residues include, e.g. , amifostine, cysteine, or other equivalent small molecule amino acid like compositions.

In certain embodiments, the FDA-approved 4-phenylbutyric acid (PBA) is administered in combination with the nanozymes, which can not only improve the efficacy of the approach to reducing nephrotoxicity, but also improve tumor therapy for resistant metastatic melanoma or other cancers.

Nanozymes useful in the practice of certain embodiments of the invention comprise at least one block copolymer and at least one antioxidant. The block copolymer comprises at least one ionically charged polymeric segment and at least one non-ionically charged polymeric segment (e.g. , hydrophilic segment). In a particular embodiment, the block copolymer has the structure A-B or B-A. The block copolymer may also comprise more than 2 blocks. For example, the block copolymer may have the structure A-B-A, wherein B is an ionically charged polymeric segment. In a particular embodiment, the segments of the block copolymer comprise about 10 to about 500 repeating units, about 20 to about 300 repeating units, about 20 to about 250 repeating units, about 20 to about 200 repeating units, or about 20 to about 100 repeating units.

The ionically charged polymeric segment may be cationic or anionic. The ionically charged polymeric segment may be selected from, without limitation, polymethylacrylic acid and its salts, polyacrylic acid and its salts, copolymers of acrylic acid and its salts, poly(phosphate), polyamino acids (e.g. , polyglutamic acid, polyaspartic acid), polymalic acid, polylactic acid, homopolymers or copolymers or salts thereof of aspartic acid, 1 ,4-phenylenediacrylic acid, ciraconic acid, citraconic anhydride, trans-cinnamic acid, 4-hydroxy-3-methoxy cinnamic acid, p-hydroxy cinnamic acid, trans glutaconic acid, glutamic acid, itaconic acid, linoleic acid, linlenic acid, methacrylic acid, maleic acid, trans-.beta.-hydromuconic acid, trans-trans muconic acid, oleic acid, vinylsulfonic acid, vinyl phosphonic acid, vinyl benzoic acid, and vinyl glycolic acid and the like and carboxylated dextran, sulfonated dextran, heparin and the like.

Examples of polycationic segments include but are not limited to polymers and copolymers and their salts comprising units deriving from one or several monomers including, without limitation: primary, secondary and tertiary amines, each of which can be partially or completely quaternized forming quaternary ammonium salts. Examples of these monomers include, without limitation, cationic amino acids (e.g., lysine, arginine, histidine), alkyleneimines (e.g., ethyl eneimine, propyl eneimine, butileneimine, pentyleneimine, hexyleneimine, and the like), spermine, vinyl monomers (e.g. , vinylcaprolactam, vinylpyridine, and the like), acrylates and methacrylates (e.g. , Ν,Ν-dimethylaminoethyl acrylate, Ν,Ν-dimethylaminoethyl

methacrylate, Ν,Ν-diethylaminoethyl acrylate, Ν,Ν-diethylaminoethyl methacrylate, t- butylaminoethyl methacrylate, acryloxyethyltrimethyl ammonium halide, acryloxyethyl- dimethylbenzyl ammonium halide, methacrylamidopropyltrimethyl ammonium halide and the like), allyl monomers (e.g. , dimethyl diallyl ammonium chloride), aliphatic, heterocyclic or aromatic ionenes. In a particular embodiment, the ionically charged polymeric segment is cationic. In a particular embodiment, the cationic polymeric segment comprises cationic amino acids (e.g. , poly-lysine).

Examples of non-ionically charged water soluble polymeric segments include, without limitation, polyetherglycols, poly(ethylene oxide), copolymers of ethylene oxide and propylene oxide, polysaccharides, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyltriazole, N-oxide of polyvinylpyridine, N-(2-hydroxypropyl)methacrylamide (HPMA), polyortho esters,

polyglycerols, polyacrylamide, polyoxazolines, polyacroylmorpholine, and copolymers or derivatives thereof.

The nanozymes of the instant invention may be synthesized by 1 ) contacting at least one block copolymer with at least one protein, 2) contacting the complex formed between the block copolymer and protein with a cross-linker, and 3) purifying the generated nanozymes from the non cross-linked components. The term "cross-linker" refers to a molecule capable of forming a covalent linkage between compounds (e.g., polymer and protein). In a particular embodiment, the cross-linker forms covalent linkages (e.g. , an amide bond) between amino groups of the ionically charged polymeric segment and carboxylic groups of the protein. In a particular embodiment, the cross-linker forms covalent linkages between amino groups of the ionically charged polymeric segment and amino groups of the protein. Cross-linkers are well known in the art. In a particular embodiment, the cross-linker is a titrimetric cross-linking reagent. The cross- linker may be a bifunctional, trifunctional, or multifunctional cross-linking reagent. Examples of cross-linkers are provided in U.S. Pat. No. 7,332,527. The cross-linker may be cleavable or biodegradable or it may be non-biodegradable or uncleavable under physiological conditions. In a particular embodiment, the cross-linker comprises a bond, which may be cleaved in response to chemical stimuli (e.g. , a disulfide bond that is degraded in the presence of intracellular glutathione). The cross-linkers may also be sensitive to pH (e.g., low pH). In a particular embodiment, the cross-linker is selected from the group consisting of linkers 3,3'- dithiobis(sulfosuccinimidylpropionate) (DTSSP) and bis(sulfosuccinimidyl)suberate (BS.sup.3). l -Ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and (N- hydroxysulfosuccinimide) may also be used for cross-linking reactions. Certain synthetic methods are described herein with respect to PEI-based nanozymes, PLL-based nanozymes, and DET-based nanozymes. In order to minimize undesired cross-linking with the amino groups of the protein, excess ionic (cationic) polymer can be used in the cross-linking reaction. In a particular embodiment, the molar ratio of crosslinker to the ionically charged polymeric segment is less than about 1.0, less than about 0.8, or less than about 0.5. In a particular embodiment, the molar ratio is about 0.5.

After synthesis, the nanozymes of the instant invention are purified from non cross- linked components. The nanozymes may be purified by methods known in the art. For example, the nanozymes may be purified by size exclusion chromatography (e.g. , using a Sephacryl.TM. S-400 column or equivalent thereof) and/or centrifugal filtration (e.g. , using a 100 kDa or 1000 kDa molecular weight cutoff). In a particular embodiment, the nanozymes are purified such that at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more of undesired components are removed from the sample. In a particular embodiment, the nanozymes are purified such that the polydispersity index (PDI) of the preparation is less than about 0.1, less than about 0.8, or less than about 0.05. In a particular embodiment, the purified nanozymes have a diameter of less than about 100 ran.

The instant invention encompasses compositions comprising at least one nanozyme that comprises an antioxidant (e.g. , a purified nanozyme), a radionuclide based agent useful, e.g., for treating cancer, and at least one pharmaceutically acceptable carrier. The compositions of the instant invention may further comprise other therapeutic agents.

While the instant invention generally describes the use of antioxidants in the nanozymes, it is also within the scope of the instant invention to use other therapeutic agents or compounds of interest in the nanozymes. The compound(s) can be, without limitation, a biological agent, detectable agents (e.g., imaging agents or contrast agents), or therapeutic agent. Such agents or compounds include, without limitation, polypeptides, peptides, glycoproteins, nucleic acids (DNA, R A, oligonucleotides, plasmids, siR A, etc.), synthetic and natural drugs,

polysaccharides, small molecules, lipids, and the like. In a particular embodiment, the protein or compound has an opposite charge (e.g., overall charge) opposite to the ionically charged polymeric segment.

In a particular embodiment, the antioxidant of the nanozyme is superoxide dismutase (SOD; e.g., copper zinc SOD or SODl) and/or catalase. For simplicity, the nanozyme is referred to as containing SOD, but the nanozymes may contain catalase. The antioxidant enzyme superoxide dismutase (SOD), particularly, SODl (also called Cu/Zn SOD) is known to catalyze the dismutation of superoxide. Thus, SOD, e.g. , SODl , can be used in antioxidant therapy.

In a particular embodiment, the methods of the instant invention comprise the administration of at least one nanozyme comprising SOD and at least one nanozyme comprising catalase. The SOD (e.g., SODl ) and catalase nanozymes may be administered as a singular composition (e.g. , with at least one pharmaceutically acceptable carrier) or administered in separate compositions (e.g. , with each composition having at least one pharmaceutically acceptable carrier). When the compositions are separate, the SOD and catalase nanozymes may be administered sequentially or simultaneously.

As used herein, the term "antioxidant" refers to compounds that neutralize the activity of reactive oxygen species or inhibit the cellular damage done by the reactive species or their reactive byproducts or metabolites. The term "antioxidant" may also refer to compounds that inhibit, prevent, reduce or ameliorate oxidative reactions. Examples of antioxidants include, without limitation, antioxidant enzymes (e.g., SOD, catalase or other enzyme with antioxidant or other enzymatic activity that can provide detoxification actions in the kidneys), sulfhydryl containing molecules, amifostine, vitamin E, vitamin C, ascorbyl palmitate, vitamin A, carotenoids, beta carotene, retinoids, xanthophylls, lutein, zeaxanthin, flavones, isoflavones, flavanones, flavonols, catechins, ginkgolides, anthocyanidins, proanthocyanidins, carnosol, carnosic acid, organosulfur compounds, allylcysteine, alliin, allicin, lipoic acid, omega-3 fatty acids, eicosapentaeneoic acid (EPA), docosahexaeneoic acid (DHA), tryptophan, arginine, isothiocyanates, quinones, ubiquinols, butylated hydroxytoluene (BHT), butylated

hydroxyanisole (BHA), super-oxide dismutase mimetic (SODm), and coenzymes-Q.

The terms "reactive oxygen species," or "oxidative species," as used herein, refer to oxygen derivatives from oxygen metabolism or the transfer of electrons, resulting in the formation of "free radicals" (e.g., superoxide anion or hydroxyl radicals).

In a particular embodiment of the instant invention, the nanozymes include therapeutic molecules, e.g., antibodies or proteins. In a particular embodiment, the protein is an antioxidant and/or a scavenger of reactive oxygen species (ROS). As described herein, the nanozymes specifically accumulate in the kidney. As such, therapeutic molecules can be delivered specifically to the kidneys using the nanozymes. Examples of specific proteins include, without limitation, superoxide dismutase (SOD) or catalase (e.g., of mammalian, particularly human, origin), cytokines, leptin (Zhang et al. (1994) Nature, 372:425-432; Ahima et al. (1996) Nature, 382:250-252; Friedman and Halaas (1998) Nature, 395:763-770), enkephalin, growth factors (e.g., epidermal growth factor (EGF; Ferrari et al. (1990) Adv Exp Med Biol. 265:93-99), basic fibroblast growth factor (bFGF; Ferrari et al. (1991) J Neurosci Res. 30:493-497), nerve growth factor (NGF; Koliatsos et al. (1991) Ann Neurol. 30:831-840)), amyloid beta binders (e.g.

antibodies), modulators of .alpha.-, .beta.-, and/or .gamma.-secretases, glial-derived

neutrotrophic factor (GDNF; Schapira, A. H. (2003) Neurology 61 :S56-63), vasoactive intestinal peptide (Dogrukol-Ak et al. (2003) Peptides 24:437-444), acid alpha-glucosidase (GAA;

Amalfitano et al. (2001 ) Genet Med. 3:132-138), acid sphingomyelinase (Simonaro et al. (2002) Am J Hum Genet. 71 : 1413-1419), iduronate-2-sultatase (I2S; Muenzer et al. (2002) Acta Paediatr Suppl. 91 :98-99), .alpha. -L-iduronidase (IDU; Wraith et al. (2004) J Pediatr. 144:581 - 588), 3-hexosaminidase A (HexA; Wicklow et al. (2004) Am J Med Genet. 127A: 158-166), acid .beta.-glucocerebrosidase (Grabowski, G. A., (2004) J Pediatr. 144:S15-19), N- acetylgalactosamine-4-sulfatase (Auclair et al. (2003) Mol Genet Metab. 78:163-174), and .alpha.-galactosidase A (Przybylska et al. (2004) J Gene Med. 6:85-92).

In certain embodiments, the radionuclide therapy comprises the use of Pb-203 DOTA- VMT-MCR1. Further, certain embodiments of the invention provide the use and compositions the comprise combinations of the nanozymes described herein with Pb-203 DOTA-VMT- MCR1.

(

The potential for RTRT to improve outcomes for cancer patients has long been recognized. ^1"6 However, most RTRT agents (e.g. , peptides) are primarily cleared by glomerular filtration and efficient reabsorption by non-specific receptors (megalin; cubulin) and as a result, nephrotoxicity (together with marrow) ¹⁵ ' ¹⁶ ' ^19-23 is a primary dose-limiting factor for RTRT. ⁶ ' ^8-16 Published approaches to overcoming dose-limiting nephrotoxicity include modifications to ligand desig n; ²⁴ - ³⁷ use of radionuclides with shorter-range emissions (e.g. , alpha-particles); and competitive inhibition of ligand accumulation in tubules by co-infusion of amino acids; albumin fragments; and succinylated gelatin. ^19"22 ' ^38"45 Co-infusion of basic amino acids (Lys, Arg) is the current standard for peptide RTRT. Most recently, chemical antioxidant amifostine and

"PEGylated" amifostine ^{46' 47} have received considerable attention, but do not specifically target kidneys; ^48"59 improvement of histological damage is questionable; ¹⁰ ' ⁶⁰ ' ⁶¹ and co-injection of amifostine has reduced radioligand tumor uptake by 48% in mice. ⁶² In addition, emerging- published evidence is demonstrating that bolus infusion of competitive inhibitors leads to ER- stress-mediated tubular-cell apoptosis and renal lesions. ⁶⁵ Thus, bolus blocking is emerging as an insult to tubules that can lead to fibrosis. In addition, emerging evidence suggests that the mechanism of amifostine kidney protection is mediated by active enzymes, ⁶⁴ but current approaches do not deliver enzymes to the kidneys. The instant invention nanozymes, alone or in combination with PBA, is the only approach that directs slow-release competitive inhibitors (e.g. , Lys residues) and active-antioxidant enzymes specifically to the glomeruli and tubules to block radioligand reabsorption and to actively reduce oxidative- and ER-stresses that are the mechanism of kidney damage. The present approach is further innovative because of the combination of nanozymes with FDA-approved PBA to reduce ER-stress-induced tubular-cell apoptosis and associated damage in kidney tubules. Importantly, PBA is an FDA-approved drug prescribed at high doses (e.g., 20g/day) ^6;> to patients with low side effects that published studies demonstrate prevents ER-stress induced tubular fibrosis. ¹⁷ ' ¹⁸ Surprisingly, data also demonstrate that nanozyme co-injection can improve peptide tumor targeting, presumably by transient disruption of glomerular filtration.

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Patent Applications

Schultz MK; Kapoor S, Burnett-Simons A. Compositions and Methods for Treating Cancer. US62/24L379; PCTUS2015/030856.

In certain embodiments, nanozymes can be co-administered with treatments for cancer, e.g. , whose side effects include disruption of or degradation of kidney function. Examples of cancer drugs in which kidney function is potentially degraded are numerous and include vemurafenib, mitomycin, gemcitabine, antiangiogenesis drugs, and many other forms of cancer therapies. In certain embodiments, the invention can be used to direct antioxidant enzymes to the kidneys to reduce damage from anticancer drugs. Certain embodiments of the invention are directed to the use of the nanozymes described herein to treat cancer, e.g. , in combination with a radionuclide therapy. In certain embodiments, the cancer is melanoma. In certain embodiments, the cancer is neuroendocrine tumors, prostate cancer, breast cancer, colon cancer, stomach cancer, glioblastoma, or any other cancer type in which anticancer drugs are administered that cause various forms of degradation of kidney function, such as proteinuria. Other cancers that are treated with anticancer drugs that can cause degradation of kidney function for which the present invention can be used to reduce the kidney damage caused by the anticancer agent include:

Acute granulocytic leukemia (see Leukemia)

Acute lymphocytic leukemia (ALL) (see Leukemia)

Acute myelogenous leukemia (AML) (see Leukemia) Adenocarcinoma (see Lung cancer)

Adenosarcoma (see Lung cancer)

Adrenal cancer

Adrenocortical carcinoma (see Adrenal cancer)

Anal cancer

Anaplastic astrocytoma (see Brain cancer)

Angiosarcoma (see Soft tissue sarcoma)

Appendix cancer

Astrocytoma (see Brain cancer)

Basal cell carcinoma (see Skin cancer)

B-Cell lymphoma (see Non-Hodgkin lymphoma (NHL))

Bile duct cancer

Bladder cancer

Bone cancer

Bowel cancer (see Colorectal cancer)

Brain cancer

Brain stem glioma (see Brain cancer)

Brain tumor (see Brain cancer) Breast cancer

Carcinoid tumors

Cervical cancer

Cholangiocarcinoma (see Bile duct cancer)

Chondrosarcoma (see Bone cancer)

Chronic lymphocytic leukemia (CLL) (see Leukemia)

Chronic myelogenous leukemia (CML) (see Leukemia)

Colon cancer (see Colorectal cancer)

Colorectal cancer

Craniopharyngioma (see Brain cancer)

Cutaneous lymphoma (see Skin cancer)

Cutaneous melanoma (see Melanoma)

Diffuse astrocytoma (see Brain cancer)

Ductal carcinoma in situ (DCIS) (see Breast cancer)

Endometrial cancer (see Uterine cancer)

Ependymoma (see Brain cancer)

Epithelioid sarcoma (see Soft tissue sarcoma)

Esophageal cancer

Ewing sarcoma (see Bone cancer)

Extrahepatic bile duct cancer (see Bile duct cancer)

Eye cancer

Fallopian tube cancer (see Ovarian cancer)

Fibrosarcoma (see Soft tissue sarcoma)

Gallbladder cancer

Gastric cancer (see Stomach cancer) Gastrointestinal cancer

Gastrointestinal carcinoid cancer

Gastrointestinal stromal tumors (GIST)

General

Germ cell tumor (see Brain cancer)

Glioblastoma multiforme (GBM) (see Brain cancer)

Glioma (see Brain cancer)

Hairy cell leukemia (see Leukemia)

Head and neck cancer

Hemangioendothelioma

Hodgkin lymphoma

Hodgkin's disease (see Hodgkin lymphoma )

Hodgkin' s lymphoma (see Hodgkin lymphoma ) Hypopharyngeal cancer (see Throat cancer)

Infiltrating ductal carcinoma (IDC) (see Breast cancer) Infiltrating lobular carcinoma (ILC) (see Breast cancer) Inflammatory breast cancer (IBC) (see Breast cancer) Intestinal Cancer

Intrahepatic bile duct cancer (see Bile duct cancer) Invasive / infiltrating breast cancer (see Breast cancer) Islet cell cancer (see Pancreatic cancer)

Jaw cancer (see Oral cancer)

Kaposi sarcoma (see Oral cancer)

Kidney cancer

Laryngeal cancer (see Throat cancer) Leiomyosarcoma (see Soft tissue sarcoma)

Leptomeningeal metastases

Leukemia

Lip cancer (see Oral cancer)

Liposarcoma (see Soft tissue sarcoma)

Liver cancer

Lobular carcinoma in situ (see Breast cancer) Low-grade astrocytoma (see Brain cancer) Lung cancer

Lymph node cancer (see Non-Hodgkin lymphoma Lymphoma (see Non-Hodgkin lymphoma (NHL))

Male breast cancer (see Breast cancer)

Medullary carcinoma (see Breast cancer)

Medulloblastoma (see Brain cancer)

Melanoma

Meningioma (see Brain cancer)

Merkel cell carcinoma (see Skin cancer)

Mesenchymal chondrosarcoma (see Bone cancer)

Mesenchymous

Mesothelioma

Metastatic breast cancer (see Breast cancer) Metastatic melanoma (see Melanoma)

Metastatic squamous neck cancer

Mixed gliomas (see Brain cancer)

Mouth cancer (see Oral cancer)

Mucinous carcinoma (see Breast cancer)

Mucosal melanoma (see Oral cancer)

Multiple myeloma Nasal cavity cancer (see Throat cancer)

Nasopharyngeal cancer (see Throat cancer)

Neck cancer (see Head and neck cancer)

Neuroblastoma

Neuroendocrine tumors (NETs)

Non-Hodgkin lymphoma (NHL)

Non-Hodgkin's lymphoma (see Non-Hodgkin lymphoma (NHL)) Non-small cell lung cancer (NSCLC) (see Lung cancer)

Oat cell cancer (see Lung cancer)

Ocular cancer

Ocular melanoma

Oligodendroglioma (see Brain cancer)

Oral cancer

Oral cavity cancer (see Oral cancer)

Oropharyngeal cancer (see Throat cancer)

Osteogenic sarcoma (see Bone cancer)

Osteosarcoma (see Bone cancer)

Ovarian cancer

Ovarian epithelial cancer (see Ovarian cancer)

Ovarian germ cell tumor (see Ovarian cancer)

Ovarian primary peritoneal carcinoma (see Ovarian cancer)

Ovarian sex cord stromal tumor (see Ovarian cancer)

Paget's disease (see Breast cancer)

Pancreatic cancer

Papillary carcinoma (see Breast cancer)

Paranasal sinus cancer

Parathyroid cancer (see Thyroid cancer)

Pelvic cancer Penile cancer

Peripheral nerve cancer (see Brain cancer) Peritoneal cancer (see Ovarian cancer)

Pharyngeal cancer (see Throat cancer)

Pheochromocytoma (see Adrenal cancer) Pilocytic astrocytoma (see Brain cancer)

Pineal region tumor (see Brain cancer)

Pineoblastoma

Pituitary gland cancer (see Brain cancer)

Primary central nervous system (CNS) lymphoma Prostate cancer

Rectal cancer (see Colorectal cancer)

Renal cell cancer (see Kidney cancer)

Renal pelvis cancer (see Kidney cancer)

Rhabdomyosarcoma (see Soft tissue sarcoma)

Salivary gland cancer (see Oral cancer)

Sarcoma (see Soft tissue sarcoma)

Sarcoma, bone (see Bone cancer)

Sarcoma, soft tissue

Sarcoma, uterine (see Uterine cancer)

Sinus cancer

Skin cancer

Small cell lung cancer (SCLC) (see Lung cancer) Small intestine cancer

Soft tissue sarcoma

Spinal cancer

Spinal column cancer (see Spinal cancer) Spinal cord cancer (see Spinal cancer) Spinal tumor (see Spinal cancer)

Squamous cell carcinoma (see Skin cancer)

Stomach cancer

Synovial sarcoma (see Soft tissue sarcoma)

T-cell lymphoma (see Non-Hodgkin lymphoma (NHL)) Testicular cancer

Throat cancer

Thymoma / thymic carcinoma

Thyroid cancer

Tongue cancer (see Oral cancer)

Tonsil cancer

Transitional cell cancer (see Bladder cancer)

Transitional cell cancer (see Kidney cancer)

Transitional cell cancer (see Ovarian cancer)

Triple-negative breast cancer (see Breast cancer) Tubal cancer

Tubular carcinoma (see Breast cancer)

Ureteral cancer (see Bladder cancer)

Ureteral cancer (see Kidney cancer)

Urethral cancer

Uterine adenocarcinoma (see Uterine cancer)

Uterine cancer

Uterine sarcoma (see Uterine cancer)

Vaginal cancer

Vulvar cancer The nanozymes described herein will generally be administered to a patient as a pharmaceutical preparation. The term "patient" as used herein refers to human or animal subjects. These nanozymes may be employed therapeutically, under the guidance of a physician or other healthcare professional.

The pharmaceutical preparation comprising the nanozymes may be conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. The concentration of nanozymes in the chosen medium will depend on the hydrophobic or hydrophilic nature of the medium, as well as the size, enzyme activity, and other properties of the nanozymes. Solubility limits may be easily determined by one skilled in the art.

As used herein, "pharmaceutically acceptable medium" or "carrier" includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding discussion. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the nanozyme to be administered, its use in the pharmaceutical preparation is contemplated.

The dose and dosage regimen of a nanozyme that is suitable for administration to a particular patient may be determined by a physician considering the patient's age, sex, weight, general medical condition, and the specific condition for which the nanozyme is being administered and the severity thereof. The physician may also take into account the route of administration of the nanozyme, the pharmaceutical carrier with which the nanozyme is to combined, and the nanozyme's biological activity.

Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the nanozymes of the invention may be administered intravenously.

Nanozymes may be administered by any method such as intravenous injection or intracarotid infusion into the blood stream, intranasal administration, oral administration, or by subcutaneous, intramuscular or intraperitoneal injection. Pharmaceutical preparations for injection are known in the art. If injection is selected as a method for administering the nanozymes, steps may be taken to ensure that sufficient amounts of the molecules reach their target cells (e.g., the kidney) to exert a biological effect. The lipophilicity of the nanozymes, or the pharmaceutical preparation in which they are delivered, may have to be increased so that the molecules can arrive at their target location. Furthermore, the nanozymes may have to be delivered in a cell -targeting carrier so that sufficient numbers of molecules will reach the target cells. Methods for increasing the lipophilicity of a molecule are known in the art.

Pharmaceutical compositions containing a nanozyme as an active ingredient in admixture with a pharmaceutical carrier can be prepared according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. , intravenous, intranasal, oral, direct injection, intracranial, and intravitreal. In preparing the nanozyme in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid

preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form in which solid pharmaceutical carriers are employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. Additionally, the nanozyme of the instant invention may be administered in a slow- release matrix. For example, the nanozyme may be administered in a gel comprising

unconjugated poloxamers.

A pharmaceutical preparation useful in the practice of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art. Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.

The appropriate dosage unit for the administration of nanozymes may be determined by evaluating the toxicity of the molecules in animal models. Various concentrations of nanozyme pharmaceutical preparations may be administered to mice, and the minimal and maximal dosages may be determined based on the beneficial results and side effects observed as a result of the treatment. Appropriate dosage unit may also be determined by assessing the efficacy of the nanozymes treatment in combination with other standard drugs. The dosage units of nanozymes may be determined individually or in combination with each treatment according to the effect detected.

The pharmaceutical preparation comprising the nanozymes may be administered at appropriate intervals, for example, at least twice a day or more until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient. The pharmaceutical preparation comprising the nanozymes may be administered in combination with at least one other therapeutic treatment (e.g., a radionuclide based therapy for cancer treatment), e.g., prior to administration of the other treatment(s), concurrently with

administration of the other treatment(s), and/or subsequent to administration of the other treatment(s).

Certain embodiments of the invention will now be illustrated by the following non- limiting Examples. Exemplary Nanozymes

Examples of nanozymes useful in the practice of certain embodiments of the invention are described hereinbelow. These include PEI-based nanozymes, PLL-based nanozymes, and DET-based nanozymes. For examples of useful methods for preparing nanozymes, please see Rosenbaugh et al, Biomaterials, 31, 5218-5226 (2010), Manickam et al. Journal of Controlled Release, 162, 636-645 (2012), Jiang et al , SOD1 nanozyme with reduced toxicity and MPS accumulation, J Control Release. 2016 Feb 27. pii: S0168-3659(16)30103-l . doi: 10.1016/j jconrel.2016.02.038, and US 2014/0120075. Other nanozyme formulations that the present invention include are PEG-poly-amino acid, poly-amifostine, poly-cysteine, or other poly-sulfhydryl or small-molecule-containing chains combined with another polymer such as PLGA or other to create the wrapper that encapsulates an active enzyme or other therapeutic designed to provide therapy to the kidneys.

PEI-based nanozyme

PEI-based nanozymes can be prepared by mixing purified poly(ethyleneglycol)-b- poly(ethyleneimine) (PEG-PEI) polymer and SODl protein at a ratio of 18.4 mg PEG-PEI to 1 mg SODl protein in 1 mL of media (pH 7.4). The mixture is then incubated for 30 min to allow for formation of complexes. SODl protein and PEG-PEI self-assemble into polyion complexes with a PEG corona and a PEI core electrostatically bound to SODl through the positively charged amine groups of the polymer and the negatively charged carboxyl groups of the protein. For additional information, please also see Rosenbaugh el al , Biomaterials, 31, 5218-5226 (2010).

Scheme 1, PEG-PEI polymer

PLL-based nanozyme

SODl and poly(ethylene glycol)-6-poly(L-lysine) (PEG-PLL) polymer stock solutions are prepared in 10 mM HEPES (pH 7.4). Non-cross-linked nanozymes (Z+ _/ -=2) are prepared as described in the PEI-based nanozyme example. Targeted degree of cross-linking are defined as the molar ratio between cross-linker 3,3 ^' -dithiobis(sulfosuccinimidyl propionate) (DTSSP) and PLL amines. Pre-calculated amount of the respective cross-linker are dissolved in the reaction buffer, quickly added to non-cross-linked nanozymes, and the reaction mixture are briefly vortexed and incubated for 2 h on ice. Unreacted cross-linker can be desalted using NAP™ columns following manufacturer's instructions. Crosslinked Nanozymes are then purified using size exclusion chromatography (SEC) (small/intermediate scale) or centrifugal filtration (large scale). SEC can be carried out using an AKTA™ Fast Protein Liquid Chromatography (FPLC) (Amersham Biosciences, Piscatawav, NJ) system. In large-scale preparations, nanozymes can be purified by centrifugal filtration using Macrosep™ Centrifugal Device (Pall Life Sciences, Ann Arbor, MI) with a MWCO of 100 kDa. Briefly, unreacted DTSSP in cross-linked nanozyme are desalted using NAP™ columns and eluate collected in 10 mM HEPES containing 0.3 M NaCl (pH 7.4). Samples are loaded onto the centrifugal device and concentrated to 10% original volume by centrifuging at 4500 rpm. Two rounds of purification can be done in 10 mM HEPES buffer containing 0.3 M NaCl (pH 7.4). For additional information, please also see Manickam et al. Journal of Controlled Release, 162. 636-645 (2012).

Scheme 2, PEG-PLL polymer

DET-based nanozymes

DET-based SOD1 nanozyme are synthesized with poly(ethylene glycol)- >-poly(aspartate diethyltriamine) (PEG-PAsp(DET)) at the polycation to SOD1 charge (at pH=7.4) ratio Z ₊ =l . DTSSP can be used as a cross-linker at stoichiometric ratio to the amount of primary amine groups on the polycation as described in the PEI-based nanozyme example. After synthesis and desalting (to remove unreacted DTSSP), nanozymes are purified to remove any unconjugated proteins and polymers. For the purification, nanozymes in 10 mM HEPES buffer (pH=7.4) containing 0.15 M NaCl are loaded onto Macrosep centrifugal devices (Pall Corporation, Port Washington, NY) and concentrated twice to about 10% of its initial volume by centrifugation at 4500 rpm at 4C. For additional information, please also see Jiang et al, SOD1 nanozyme with reduced toxicity and MPS accumulation, J Control Release. 2016 Feb 27. pii: S0168- 3659(16)30103-1. doi: 10.1016/j.jconrel.2016.02.038.

Scheme 3, PEG-PAsp(DET) polymer

Examples.

Example 1. Nanozymes loaded with Cu-Zn superoxide dismutase accumulate preferentially in mouse kidneys in naive mice. SCID hairless mice were injected with 1-125 labeled nanozymes that had been preloaded with active superoxide dismutase and the biodistribution of the nanozymes was determined at 0.5, 1.0, and 2.0 hours post tail vein injection. The nanozymes preferentially accumulate in the kidneys and nearly 70% injected dose per gram (%ID/g) of kidney tissue was observed in these studies. n=3 mice at each timepoint. A small amount of material is observed to remain in the blood (<10%ID/g) and the material appears to be clearing through the renal system into the bladder. These results indicate that the antioxidant loaded nanozymes can reduce kidney toxicity that arises from radiation localized to the kidneys, e.g., in peptide-targeted radionuclide therapy for cancer. These results are presented in Figure 3.

Example 2. Nanozymes loaded with Cu-Zn superoxide dismutase accumulate preferentially in mouse kidneys in mice bearing melanoma tumors. Melanoma tumors were induced subcutaneously on the shoulder of SCID hairless mice and allowed to grow to approximately 125-250 mm . Mice were injected with 1-125 labeled nanozymes that had been preloaded with active superoxide dismutase and the biodistribution of the nanozymes was determined at 0.5, 1.0, and 2.0 hours post tail vein injection. In these studies, the nanozymes preferentially accumulated in the kidneys and nearly 50% injected dose per gram (%ID/g) of kidney tissue was observed. n=3 mice at each time point. The urine and bladder accumulation was established together also for these studies. A small amount of material is observed to remain in the blood (<10%ID/g) and the material appears to be clearing through the renal system into the bladder. Importantly, very little accumulation of the nanozymes was observed in the tumors that had been induced in the mice. These results indicate that the antioxidant loaded nanozymes reduce kidney toxicity that arises from radiation localized to the kidneys in peptide-targeted radionuclide therapy for cancer. These results are presented in Figure 4.

Example 3. Figure 5 provides data indicating that nanozyme coinjection improves the biodistribution of radiopeptide [ ^ZUJ Pb]DOTA-VMT-MCRl for imaging and therapy for melanoma. In vivo biodistribution of [ 203 Pb]DOTA-VMT-MCRl peptide in melanoma tumor bearing mice with (right bars) and without (left bars) co-injection (tail vein) of nanozymes.

Tumors and organs were harvested at 3h (Figure 5 A) and 24 h (Figure 5B) post injection (n = 3; ID%/g ± SEM). At 3 h post injection, kidney retention of [ ²⁰³ Pb]DOTA-VMT-MCRl decreased by 16% when co-injected with nanozymes, while tumor accumulation increased 23%. Retained activity decreased in heart, lung, spleen, muscle, and brain with co-injection of nanozymes, but was elevated by 26% relative to [ ²⁰³ Pb]DOTA-VMT-MCRl injected without nanozymes. These data support the hypothesis that active-enzyme-encapsulating nanozymes can be designed to accumulate in kidneys to block kidney accumulation of radiopeptides and also to increase tumor radiation dose and improve therapy for cancer. All documents cited herein are incorporated by reference. While certain embodiments of invention are described, and many details have been set forth for purposes of illustration, certain of the details can be varied without departing from the basic principles of the invention.

The use of the terms "a" and "an" and "the" and similar terms in the context of describing embodiments of invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms

"comprising," "having," "including," and "containing" are to be construed as open-ended terms ( . e. , meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. In addition to the order detailed herein, the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments of invention and does not necessarily impose a limitation on the scope of the invention unless otherwise specifically recited in the claims. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.