Pyrimidine prodrugs formulations and methods of uses thereof

Abstract

The application relates to the treatment of cancer with a pyrimidine analog or prodrug thereof, pyrimidine prodrugs formulations, methods of making the formulations, and uses of these formulations for treating cancer. For example, the application relates to the antitumor activity of the prodrug 2',2'-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041) (I) in a formulation with DSPE-PEG2000-NH2 to improve the half-life of 5-aza-2',2'-difluorodeoxycytidine (NUC013) to treat cancer.

Description

[0001] PYRIMIDINE PRODRUGS FORMULATIONS AND METHODS OF USES THEREOF REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US 63 / 490.853 filed on March 17, 2023 which is hereby incorporated by reference. TECHNICAL FIELD The technical field generally relates to the treatment of cancer with a pyrimidine analog or prodrug thereof, pyrimidine prodrugs formulations, methods of making the formulations, and uses of these formulations for treating cancer. For example, the application relates to a formulation of the prodrug 2',2'-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC-041) with DSPE-PEG2000-NH2 to improve the half-life of 5-aza-2',2'-difluorodeoxycytidine (NUC013) to treat cancer. BACKGROUND Pyrimidine analogs are known to be effective chemotherapeutic agents, as DNA methyl transferase inhibitors (DNMTI) and ribonucleotide reductase inhibitors (RNRI). However, DNMTI are unstable in water and DNMTI and RNRI cytidine analogs are rapidly deaminated in circulation. One approach that has been proposed to prolong the circulatory half-life of pyrimidine analogs was to generate a prodrug that would result in a more hydrophobic molecule that could be formulated in a lipid based nanoformulation and would rapidly release the active drug upon exposure to an aqueous environment at the tumor site. There continues to exist a need for novel cancer therapeutics with improved efficacy, safety, and / or pharmacokinetic profiles. SUMMARY According to one aspect, NUC013 and its prodrug NUC041 have been shown to be immunotherapeutics, In one aspect, NUC013 and its prodrug NUC041 have been shown to decrease the proportion of necrotic cells in tumors. In one aspect, NUC013 and its prodrug NUC041 have been shown to decrease the proportion of cell expressing MAGE-A. In one aspect, NUC013 and its prodrug NUC041 have been shown to be able to treat particularly difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer. In one aspect, the present application relates to a formulation of a pyrimidine prodrug in a formulation comprising PEG. In one aspect, the present application relates to a formulation of a pyrimidine prodrug in a formulation comprising PEG, wherein the pyrimidine is 5-aza-2',2'-difluorodeoxycytidine (NUC013). In one aspect, the formulation described therein, further comprises a phospholipid. In one aspect, PEG is DSPE-PEG2000-CN (DSPE-020-CN) or DSPE-PEG2000-NH2. In one aspect, PEG is at a concentration from greater than zero to 20% weight to weight. In one aspect, the pyrimidine prodrug is 2',2'-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041) having the structure: . In one aspect, the present application describes a method of treatment of cancer with 5- aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or the formulation described therein. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or the formulation described therein for treating cancer. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or the formulation described therein for the manufacture of a medicament for treating cancer. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or the formulation described therein for affecting both arms of the anti-tumor immune system. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or the formulation described therein for the manufacture of a medicament for affecting both arms of the anti-tumor immune system. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation described therein for treating cancer. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation described therein for affecting both arms of the anti-tumor immune system. In one aspect, the present application describes a method of treating cancer by stimulating both arms, innate and adaptive, of the immune system with 5-aza-2',2'- difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the present application describes a method of treating cancer by stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the present application describes a method of treating cancer by increasing the proportion of NK and NKT cells positive for granzyme B, with 5-aza-2',2'- difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the present application describes a method of treating cancer by increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the present application describes a method of treating cancer leading to a decrease in the proportion of necrotic cells in the tumor, with 5-aza-2',2'- difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the present application describes a method of treating cancer by decreasing the proportion of cells expressing MAGE-A, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the present application describes a method of treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer, with 5- aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug. In one aspect, the pyrimidine prodrug described therein is 2',2'-difluoro-5- azadeoxycytidine-trimethylsilyl (NUC041). In one aspect, the formulation of the pyrimidine prodrug described therein is a formulation comprising PEG. In one aspect, the formulation described therein further comprises a phospholipid. In one aspect, the present application describes a formulation comprising PEG, wherein PEG is DSPE-PEG2000-CN or DSPE-PEG2000-NH2. In one aspect, the present application describes a formulation comprising PEG, wherein PEG is at a concentration from greater than zero to 20% weight to weight. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for increasing the proportion of NK and NKT cells positive for granzyme B. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for leading to a decrease in the proportion of necrotic cells in the tumor. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for decreasing the proportion of cells expressing MAGE-A. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for increasing the proportion of NK and NKT cells positive for granzyme B. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for leading to a decrease in the proportion of necrotic cells in the tumor. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for decreasing the proportion of cells expressing MAGE-A. In one aspect, the present application describes a use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer. In one aspect, the pyrimidine prodrug described therein is 2',2'-difluoro-5- azadeoxycytidine-trimethylsilyl (NUC041). In one aspect, the formulation of the pyrimidine prodrug described therein is a formulation comprising PEG. In one aspect, the formulation described therein further comprises a phospholipid. In one aspect, the present application describes a use, wherein PEG is DSPE-PEG2000- CN or DSPE-PEG2000-NH2.In one aspect, the present application describes a use, wherein PEG is at a concentration from greater than zero to 20% weight to weight. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by stimulating both arms, innate and adaptive, of the immune system. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by increasing the proportion of NK and NKT cells positive for granzyme B. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer leading to a decrease in the proportion of necrotic cells in the tumor. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by decreasing the proportion of cells expressing MAGE-A. In one aspect, the present application describes 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer. In one aspect, the present application describes a 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use, wherein the pyrimidine prodrug is 2',2'-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041). In one aspect, the present application describes a 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use, wherein the formulation of the pyrimidine prodrug is a formulation comprising PEG. In one aspect, the present application describes a 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use, wherein the formulation further comprises a phospholipid. In one aspect, the present application describes a 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use, wherein PEG is DSPE-PEG2000-CN or DSPE-PEG2000-NH2. In one aspect, the present application describes a 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use, wherein PEG is at a concentration from greater than zero to 20% weight to weight. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Plot of % PEG (x-axis) vs AUC (ng*h / mL). Data from Table 2. Figure 2: Plot of % PEG (x-axis) vs AUC (ng*h / mL). Data from Table 3. Figure 3: Plot of % PEG (x-axis) vs AUC (ng*h / mL). Data from Table 4. Figure 4: (A): Mice with 786-O tumors were administered 0.56 mg NUC041 SC in FS-14 qod or PBS (n =10 per group). Graph of 786-O growth in NUC041 treated versus vehicle control animals. Mean tumor volume at study initiation was 111 mm3. (B, C): Mice with Caki-1 tumors were administered 0.56 mg NUC041 SC in FS-14 qod or PBS (n =10 per group). Mean tumor volume at study initiation was 98 mm3. (B): Graph of Caki-1 growth in NUC041 treated versus control animals. (C): Survival proportion in Caki-1 implants, treated versus control animals. (D): Mice with Caki-1 tumors were administered 0.8 mg NUC041 SC in FS-19 qod or PBS (n =10 per group). Graph of Caki-1 growth in NUC041 treated versus control animals. Mean tumor volume at study initiation was 66 mm3. Figure 5: (A): Mice with CFPAC-1 tumors were initially administered 1.2 mg NUC041 SC in FS-19 qod or PBS (n =10 per group). Graph of CFPAC-1 growth in NUC041 treated versus vehicle control animals. Mean tumor volume at study initiation was 61 mm3. (B, C): Mice with SW1990 tumors were administered 0.8 mg NUC041 SC in FS-19 qod or PBS (n =10 per group). Mean tumor volume at study initiation was 65 mm3. (B): Graph of SW1990 growth in NUC041 treated versus control animals. (C): Survival proportions in SW1990 implants, treated versus control. Figure 6: Triple negative breast cancer xenograft: (A, B): Mice with DU4475 tumors were initially administered 0.8 mg or 0.6 mg NUC041 SC in FS-19 qod or PBS (n =10 per group). Mean tumor volume at study initiation was 57 mm3. (A): Graph of DU4475 growth in NUC041 treated versus vehicle control animals. (B): Survival proportions in DU4475 implants, treated versus control. Figure 7 (A, B): Representative images of tumors from all 10 vehicle control mice inoculated with Caki-1 cells at the end of the study. Hematoxylin and eosin (H&E) staining at 200X. Figure 8 (A, B): Representative images of tumors from all 10 NUC041 treated mice inoculated with Caki-1 cells at the end of the study. Hematoxylin and eosin (H&E) staining at 200X. DETAILED DESCRIPTION General Definitions All technical and scientific terms used herein have the same meaning as commonly understood by one ordinary skilled in the art to which the present technology pertains. For convenience, the meaning of certain terms and phrases used herein are provided below. To the extent the definitions of terms in the publications, patents, and patent applications incorporated herein by reference are contrary to the definitions set forth in this specification, the definitions in this specification control. The section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter disclosed. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, the singular forms "a", "an", and "the" include plural forms as well, unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" also contemplates a mixture of two or more compounds. It should also be noted that the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. Furthermore, to the extent that the terms “including”, "includes", "having", "has", "with", or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising”. The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in treatment, healing, prevention, or amelioration of a disorder, disorder, or side effect, or a decrease in the rate of advancement of a disorder or disorder. The term also includes within its scope amounts that are effective to enhance normal physiological function. As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disorder or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. The terms "patient” or “subject" as used herein generally refer to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be either a patient or a healthy human. The expression "pharmaceutically acceptable carrier, diluent, or excipient" and equivalent expressions, refer to a non-toxic carrier, diluent, or excipient that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, diluents or excipients that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. The term “cancer” refers to a cellular proliferative disease. It includes hematologic cancers, such as leukemia, lymphoma, and myeloma, and nonhematologic cancers, such as solid tumor carcinomas (e.g., skin, breast, ovarian, cervix, prostate, pancreas, stomach, colon, colorectal, lung, liver, kidney, bladder, thyroid), sarcomas, and gliomas. In one aspect, cancer includes kidney tumor, ductal adenocarcinoma, pancreatic adenocarcinoma, breast (TNBC) cancer, renal cell carcinoma RCC, and / or pancreatic cancer. Abbreviations may also be used throughout the application, unless otherwise noted, such abbreviations are intended to have the meaning generally understood by the field. Of note: qod every other day S t ti / th t hi h i h Compounds The compounds of the present application may be prepared by conventional chemical synthesis. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction duration, etc. delineated herein are for purposes of illustration only and one of ordinary skill in the art will recognize that variation of the reaction conditions can produce the desired products of the present description. Synthetic chemistry transformations and / or protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art. The synthesized compounds can be separated from a reaction mixture and further purified by standard methods such as column chromatography, high pressure liquid chromatography, or recrystallization. The compounds of the present description may be modified by appending various functionalities via any synthetic means delineated herein to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion. The central feature of cancer is increasingly considered to be an unstable and disrupted epigenome, i.e., the epigenetic information in a cell, comprising DNA methylation, post- translational modifications of histones, and higher-order chromatin structure1. DNA methyltransferase (DNMT) inhibitors are a class of drugs used in the treatment of cancer that aim to express aberrantly silenced genes through hypermethylation, e.g., genes associated with reduced proliferation, cell differentiation, apoptosis, and senescence. They include pyrimidine analogs. Decitabine (5-azadeoxycytidine) and 5-azacytidine (5- azaC) remain the only approved drugs of this class but suffer from relative toxicity and poor stability in water2. The structure of 5-azadeoxycytidine (decitabine) and 5-azacytidine (5-azaC) is NUC013 (5-aza-2',2'-difluorodeoxycytidine) is a DNA methyl transferase inhibitor (DNMTI) and ribonucleotide reductase inhibitor (RNRI) described in US 9493500 (Daifuku).3It has been shown to be significantly safer and more effective than decitabine in xenograft models of human leukemia and colon cancer. However, it suffers from a similar short half- life as other DNA methyltransferase inhibitors with a 5-azacytosine base, which is problematic for nucleosides that primarily target tumor cells in S phase.4The structure of 2',2'-difluoro-5-azadeoxycytidine (NUC013) is . Because of the relative instability of 5-azanucleosides, a prodrug approach was developed for packaging in a hydrophobic vehicle to protect NUC013 from hydrolysis and deamination to improve the pharmacology of NUC013. NUC013 was conjugated with trimethylsilanol (TMS) at the 3′ and 5′ position of the sugar, rendering the molecule hydrophobic and producing 3′,5′-di-trimethylsilyl-2′,2′-difluoro-5-azadeoxycytidine (NUC041). NUC041 was designed to be formulated in a hydrophobic (i.e. lipid) vehicle, protecting it from deamination and hydrolysis (US 10 479 807 (Daifuku)).5In contact with water or blood, the trimethylsilyl (TMS) moieties are readily hydrolyzed to release the active molecule NUC013. The half-life of NUC013 administered intravenously in mice is 20.1 min, while that of NUC013 derived from intramuscular NUC041 formulated in a pegylated- phospholipid depot is 3.4 h.6The structure of 2’,2’-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041) is: In some embodiments, or can be treated using the compounds or their pharmaceutically acceptable salts, solvates, or prodrugs, and / or formulations described herein include cancer. In some embodiments, the therapeutically effective amount of a compound as defined herein can be administered to a patient or subject, alone, in combination or admixed with a pharmaceutically acceptable carrier, diluent, or excipient and / or other compounds for treating cancer. Compositions and formulations described herein may be administered orally, parenterally, by inhalation spray, by injection, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Compositions described herein may be administered parenterally. The term “parenteral” as used herein includes subcutaneous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Other modes of administration also include intradermal or transdermal administration. Formulation of a compound or a composition is the multistep pharmaceutical process where the active drug is mixed with other components by considering at least the factors of particle size, polymorphism, pH, and solubility, as all of these can influence bioavailability and hence the activity of a drug, and becomes the final beneficial medicinal product. Benefits and constraints of the active pharmaceutical ingredients (APIs), valuable excipients, associated interactions, and manufacturing procedure are the main basic components for a successful pharmaceutical formulation. Besides the knowledge of different formulation parameters, it is significant to understand the toxicology, pharmacokinetics, chemical, and physical characterization. The clinical relevance of pharmaceutical formulations is that they have a significant impact on one’s quality of life, disease outcomes, and adherence to the treatment protocol. Formulations can include dosage forms. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, depots, syrups and elixirs. An emulsion is a mixture of two or more liquids that are normally immiscible (unmixable or unblendable) owing to liquid-liquid phase separation. Are usually oil and water emulsions. They will vary upon their oil-to-water ratios, other additives, and their intended route of administration. They can be topical dosage forms, to be used on the surface of the skin, transdermally, ophthalmically, rectally, or vaginally, or highly liquid emulsion to be used orally, or may be injected. Microemulsions are for example nanoemulsions of soybean oil, with particles that are 400– 600 nm in diameter. The process is mechanical. The smaller the droplet the greater the surface tension and thus the greater the force required to merge with other lipids. The oil is emulsified with detergents using a high-shear mixer to stabilize the emulsion so, when they encounter the lipids in the cell membrane, they force the lipids to merge with themselves. A solution is a homogenous mixture of two or more components, i.e. a solute dissolved in a solvent such as water or an organic solvent. Pharmaceutical solutions include solutes such as drugs and excipients, dissolved in a liquid solvent such as water. Non-aqueous solvent systems which contain solvents other than water, either alone or in addition to water, are used when the drug is insufficiently soluble or stable in aqueous systems, or when a solution is intended for specific properties, such as sustained drug absorption, however the solvent must not be toxic for the patient. A pharmaceutical suspension is a biphasic liquid or semi-solid dosage form where the finely divided insoluble solid drug particles are dispersed in a liquid or semi-solid medium. The insoluble solid drug particles act as the dispersed phase or internal phase. Based on method of preparation, suspensions can be divided into two categories, flocculated and deflocculated systems. A syrup is a pharmaceutical solution that completely dissolves into its solvent, with the distribution of drugs within the solution being even. Syrup is concentrated, viscous, aqueous solution of sugar or a sugar substitute, with or without flavor, and medical substance. An elixir is a sweet liquid containing at least one active ingredient designed to be taken orally. Nanoformulations refer to drug delivery through nanomedicine. Their efficiency is based upon: a) efficient encapsulation of the drugs, b) successful delivery of drug to the targeted region of the body, and c) successful release of the drug. Drug delivery vehicles represent different ways that medications can be packaged so that the drug can safely travel within the body. Some common examples of drug delivery vehicles include micelles, liposomes, or nanoparticles. In one aspect, the delivery vehicle comprises a lipid. Non-limiting examples include a cationic lipid, a neutral lipid, an anionic lipid, a helper lipid, a stealth lipid, and / or a polyethylene glycol (PEG) lipid. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include excipients such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable carriers and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. 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 medium prior to use. In order to prolong the effect of a provided compound, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil carrier. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues. Dosage forms for topical or transdermal administration of a compound of the present description include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of the present description. Additionally, the description contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. Pharmaceutically acceptable compositions and formulations provided herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promotors to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. The amount of provided compounds that may be combined with carrier materials to produce a composition in a formulation, a specific dosage form or treatment regimen will vary depending upon the patient to be treated and the particular mode of administration. It will depend upon a variety of factors, including age, body weight, general health, sex, diet, mode of administration, time of administration, rate of excretion, drug combination, the judgment of the treating physician, the disorder being treated, the severity of the particular disorder being treated, the activity of the specific compound employed; the specific composition 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. The compounds of the invention may be administered in a composition, or in a combination, sequentially or simultaneously. Compounds or compositions described herein may be administered using any amount and any route of administration effective for treating or lessening the severity of the disorders or disorders as contemplated herein. Provided compounds are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The expression “unit dosage form” as used herein refers to a physically discrete unit of agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. Pharmaceutically acceptable compositions and formulations of this disclosure can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In certain embodiments, provided compounds may be administered at dosage levels of about of between 0.01 – 100 mg / kg, 0.01 mg / kg to about 50 mg / kg and preferably from about 0.1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In one embodiment, treatment regimens according to the present description comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of the present description per day in single or multiple doses. Upon improvement of a subject’s condition, a maintenance dose of a compound, or composition of the present description may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disorder symptoms. In one aspect, the formulation described herein comprises PEG (polyethylene glycol). PEG is a polyether compound and hydrophilic compound. It is considered biologically inert and safe (it has a GRAS (generally recognized as safe) designation) and is used in a variety of products and formulations. Polyethylene glycol (PEG) is produced from polymerization of ethylene oxide, ethylene glycol, or diethylene glycol. The chemical structure of PEG is HO-[CH2-CH2-O]n-H, where n is the number of ethylene oxide units and the molecular weight is calculated by (44 g / mol)*n. PEG is hydrophilic and soluble in water, methanol, ethanol, acetonitrile, glycerin, glycols, benzene, and dichloromethane. PEG can have different geometries, including linear, tube, branched, star, and comb.10PEG is also very flexible and exhibits high chain mobility that results in a large number of polymer chain conformations. PEG can be used to form a shell around micelles. PEG shells have a hydration sheath that sterically prevents biomacromolecules from penetrating the polymer layer and binds to the core by hydrophobic or electrostatic interactions. PEG is a common product in drug formulations, pharmacological and food products. PEG units used in drug formulations and consumables generally range between molecular weights of 200–60,000 Da10. In one aspect, the PEG molecular weight is between 200-2000 Da, 200-1000 Da, 500-1000 Da, or 1000-2000 Da. Depending on the length of the polymer chain, PEG may be defined by its molecular weight: a PEG with n = 9 would have an average molecular weight of approximately 400 daltons and would be labeled PEG 400. Similarly, PEG 2000 would have an average molecular weight of approximately 2000 daltons. The molecular weight of PEG used in nanomedicines such as Doxil is 2000 Da. Conjugation of the end hydroxyl group on PEG to reactive groups on compounds to make formulations and larger PEG-matrices is also a common use for PEG, in the form of PEGylated nanoparticles or medicine that allows for improved circulation, sustained release, improved efficacy and / or dissolution of drug, or production of artificial PEG-environment. PEG is the most common component of nanomedicines, largely due to its stealth properties. Anticancer drug, doxorubicin, conjugated to PEGylated nanotubes decreased tumor size in mice. Doxil is a PEGylated liposome formulated to deliver anti- cancer drug doxorubicin, that is approved for therapy of ovarian cancer, multiple myeloma, and AIDS-related Kaposi’s sarcoma (https: / / www.rxlist.com / doxil-drug.htm#indications). One of the benefits of this liposomal formulation in comparison to doxorubicin hydrochloride is the reduction in the drug’s cardiotoxicity, that is attributed to the doxorubicin-mediated induction of inflammatory cytokines in cardiomyocytes.10Another PEGylated cancer nanomedicine, liposomal irinotecan or Onyvide, is approved for cancer therapy and has several advantages over the traditional formulation of irinotecan such as improved pharmacokinetics. Clinical use of this formulation is associated with severe neutropenia and neutropenic sepsis; it may cause severe hypersensitivity reactions, including anaphylaxis. In one aspect, PEG is combined with a lipid moiety, such as lipid or a phospholipid, such as DSPE, DMG, DSG and / or DOPE. PEG lipid has been used extensively to improve circulation times for liposome encapsulated (LNP) drugs and reduce non-specific uptakes. Examples of PEG Lipids are available at Broadpharm https: / / broadpharm.com / product- categories / lipid / peg-lipid#dope-peg-cooh and are described below: DSPE-PEG-Amine, MW 2,000 is a PEG polymer containing DSPE and amine end groups. The DSPE-PEGs have been FDA approved for medical applications. The hydrophobic properties of the DSPE allow for the encapsulation and congregation of other hydrophobic drugs. The hydrophilic PEG increases the water solubility of the overall compound allowing for the delivery of the drug. The amine group can react with activated NHS ester to form a stable amide bond. DOPE-PEG-Amine, MW 2,000 is an amphiphilic PEG polymer which can self-assemble in water to form micelles. The polymer can be used to prepare liposomes or nanoparticles for drug delivery applications. Amine moiety is reactive with NHS-ester to form an amide bond. DSPE-PEG-CH2COOH, MW 3,400 is a phospholipid polyPEG. The lipid tails can be used to encapsulate hydrophobic drugs, while the PEG chain maintains its water solubility. The polymer is useful for preparation of liposomes or other nanoparticles in drug nanocarrier applications. Carboxylic acid moiety can be reacted with amine molecule in the presence of activator, such as HATU or EDC, to form a stable amide linkage. DSPE-PEG-NHS, MW 2,000 is a phospholipid PEG polymer which can be used to encapsulate drugs / therapeutics agents in targeted drug delivery system, such as liposome based mRNA vaccine. The NHS-ester is reactive with amine at pH 6.5 to 7.5 to form a stable amide bond. Cyanur PEG-PE is an endgroup functionalized PEG-lipid conjugate. 1, 2-Distearoyl-sn- glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE- PEG) is a phospholipids- polymer conjugate composed by a hydrophobic core (DSPE) and a hydrophilic shell (PEG). US 9517202 describes phospholipid depot compositions for insulin and other drugs. In some embodiments, the at least one PEG-lipid conjugate may be selected from, but is not limited to at least one of Siglec-1L-PEG-DSPE, R)-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG- PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG- DMG, PEG-DMA, PEG-Ceramide C16, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-C11, PEG2000- PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click C10, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000- DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG- DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE- PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE- mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol- polyethyleneglycol, Cl8PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-PEG2000, ®-2,3-bis(octadecyloxy)propyl-1- (methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X. In one aspect, the % (w / w) of the PEG conjugated with a lipid moiety is from about 0.1% to about 10%, from about 1% to about 10%, from about 5% to about 10%, from about 8% to about 10%, from about 9% to about 10%. In one aspect, the % (w / w) of the PEG conjugated with a lipid moiety is about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 89 or 10%. EXAMPLES Formulations NUC013 (5-aza-2’,2’-difluorodeoxycytidine) is a DNA methyl transferase inhibitor (DNMTI) and ribonucleotide reductase inhibitor (RNRI) that is rapidly hydrolyzed in an aqueous medium. To address this issue, a prodrug, NUC041 (3’,5’-di-trimethylsilyl-2’,2’-difluoro-5- azadeoxycytidine), was developed. This prodrug was designed to be highly lipophilic, packaged in a lipid vehicle, and hence protected from hydrolysis. Upon contact with water, the trimethylsilyl (TMS) moieties are hydrolyzed and release the active molecule NUC013. Initial development was carried out with an unfilterable suspension formulation with DSPE- PEG-CN and NUC041 for intramuscular injection.7As this formulation showed some promise with the prolongation of the half-life of NUC013, a further development effort was undertaken for an emulsion or solution that would be injectable subcutaneously and could be filtered through a 0.22 µM filter. US patent 9517 202 (Latitude) describes injectable one-phase gel compositions comprising 30-60% phospholipid: 0.1-65% water: 10-50% oil. Based on prior experience and the literature, the original hypothesis was that the addition of DSPE-PEG2000 would provide a better half-life for NUC013.8,9,10To this end a number of formulations were tested with different concentrations of DSPE-PEG2000-NH2and other ingredients. Ingredients FS-1 FS-13 FS-14 FS-15 FS-16 FS-17 FS-18 FS-19 NUC041 6 6 6 6 6 6 6 6 ght . , y %. Below are the pharmacokinetics of FS-1, and FS-13 to FS-15 in mice.

[0002] % (w / w) C PE- ma AUC MRT DS x T 0-last 0-last max(h) T1 / 2(h) Formulation (ng / mL) (ng.h / mL) (h) ce. um concentration, Tmax is the time associated with Cmax, T1 / 2 is the half-life, MRT is the mean residence time and AUC is the area under the curve. SD: standard deviation. While addition of DSPE-PEG2000-NH2 to FS-1 had little impact on half-life, it did affect AUC as shown in Figure 1, with the best AUC achieved with formulation FS-14 (10% DSPE-PEG2000-NH2). Addition of DSPE-PEG2000-NH2 to FS-1 results in more consistent pharmacokinetics between animals, as measured by smaller standard deviations, in all relevant parameters, and in particular T1 / 2 and AUC, except for Tmax. Tmax appears to be unaffected probably because of the relatively infrequent sampling schedule. Another study was designed, using a lower dose of study drug that is closer to that used in animal tumor experiments. % (w / w) CmaxAUC0-lastMRT0-lastce. 3 male CD-1 mice per group, 25 µL of 60 mg NUC041 / mL. Where: Cmax is the maximum concentration, Tmax is the time associated with Cmax, T1 / 2 is the half-life, MRT is the mean residence time and AUC is the area under the curve. Figure 2 shows a best AUC achieved at a DSPE-PEG2000-NH2concentration of 9%. These results were confirmed in rats (see Table 4 and Figure 3). Formulation % (w / w) Cmax Tmax (h) T1 / 2 (h) AUC0-last MRT0-last DSPE- (ng / mL) (ng.h / mL) (h) ats. um concentration, Tmax is the time associated with Cmax, T1 / 2 is the half-life, MRT is the mean residence time and AUC is the area under the curve. In summary, adding DSPE-PEG2000-NH2 to FS-1 at a concentration of DSPE-PEG2000- NH2 of 9%. Improves the AUC of NUC013. Screening pharmacokinetics were performed on NUC041 in FS-19 on 3 dogs and demonstrated a half-life of 4.3 hours. These experiments could be performed in non-human primates and humans. These experiments could be performed with a lower percentage of DSPE-PEG2000-NH2. These experiments could be performed with a higher percentage of DSPE-PEG2000-NH2. These experiments could be performed with other PEG formulations. Methods, uses and administration The molecules and the formulations described herein can be useful for the treatment or prevention of cancer. In certain embodiments, the present description thus provides a method for treating or preventing cancer, comprising administering to a patient or subject identified as in need thereof, at least one formulation as defined herein. The identification of those patients who are in need of treatment for cancer is well within the ability and knowledge of one skilled in the art. Certain of the methods for identification of patients which are at risk of developing cancer which can be treated by the subject method are appreciated in the medical arts, such as family history, and the presence of risk factors associated with the development of that disorder state in the subject patient. A clinician skilled in the art can readily identify such candidate patients, by the use of, for example, clinical tests, physical examination and medical / family history. The molecules and the formulations described herein may prevent or treat cancers, preferably solid cancers, preferably RCC, TNBC or pancreatic cancer. The formulation described herein may be useful for improving the pharmacokinetics of NUC013 released from NUC041, where addition of PEG to FS-1 results in more consistent pharmacokinetics between animals, as measured by smaller standard deviations, and in particular T1 / 2 and AUC. The molecules and formulations described herein may be useful for: i. CD8-T cell proliferation ii. NK cell proliferation and increase in granzyme B in NKT cells iii. NKT cell proliferation and increase in granzyme B in NKT cells iv. Treg inhibition v. MDSC inhibition or vi. Targeting and treating of MAGE-A expressing tumor cells. The molecules and formulations described herein may be useful for doing all the above mentioned immunomodulatory activities simultaneously, i.e. affecting both arms of the immune system, i.e. the innate immune system and the adaptive (or acquired) immune system. The molecules and the formulations described herein may be useful for treatment of tumors expressing MAGE-A. DNMTI generally increase MAGE-A expression, so NUC013 / 041 would most likely do that as well. However a significant decrease was observed, likely due to MAGE-A being targeted by CTLs upregulated by treatment. The molecules and the formulations described herein may be useful to treat MAGE-A expressing tumors as a marker of clinical activity of NUC013 / 041, short of improved survival or remission. EXAMPLES While previously, it had been speculated that NUC013 / 041 may have stimulated an immune response mediated by NK cells,6the present research has demonstrated that this is indeed the case, but that NUC013 / 041 are responsible for an unexpected much broader antitumor immune response, affecting both arms of the anti-tumor immune system. Other therapeutics may have an effect on a specific cell type, but NUC013 / 041 has a pattern of anti-tumor activity that is unique and not heretofore described in the literature. Using the described herein formulations as vehicle for NUC041, specifically FS-14, and subsequently FS-19. A study was conducted in a syngeneic mouse model, to demonstrate in immunocompetent mice the value of NUC041 in FS-14 as an immunotherapeutic. Female BALB / c mice, weighing approximately 18-22 g, were used for the Renca syngeneic tumor model. Each mouse was inoculated subcutaneously at the right flank with tumor cells (5 x 106) in 200 µL of PBS mixed with Matrigel (50:50) for tumor development. When the tumor reached an approximate volume of 270 mm3, treatment of BALB / c female mice was initiated with of 1 mg NUC041 subcutaneously (SC) every other day (qod) for two doses or the same volume of PBS (n = 4 per group). Tumors were harvested two days after the last dose and submitted for fluorescent-activated cell sorting (FACS) analysis. · The percentage of cells positive for granzyme B in the control group was 70.1% and increased to 82.2% in the treated group (p = 0.024), while in NKT cells it increased from 49.9% to 74.2% (p = 0.004). · An increased ratio of CD8 cytotoxic T cells to Treg cells were noted in treated compared to control mice (0.69 to 2.86, p = 0.0026) and CD4 effector to Treg (3.70 to 8.74, p = 0.022). · The percentage of live cells increased for NK (0.50 to 0.78), NKT (0.062 to 0.085), and CD8-T (0.45 to 0.81), though these differences were not statistically significant. · T-regulatory (Treg) cells decreased in treated animals from 0.69 to 0.30 (p = 0.071). · The population of live tumor cells decreased from 47.1% to 32.9 (p = 0.056). · Lower populations of MDSC cells were noted in treated versus control, for PMN- MDSC (23.0% to 15.9%, p = 0.065) and M-MDSC (8.0% to 3.7%, p = 0.034). · Finally, as a marker of NUC041 activity, an analysis was performed on presence of Mage-A expression. Live tumor cells in control animals were 91.2% positive for Mage-A expression versus 64.2% (p = 0.046) in treated animals. Comparing how NUC013 / 041 performs compared to other DNMTI, namely the approved DNMTI 5-azacytidine and decitabine, and the investigational prodrug of decitabine, guadecitabine: Attribute Current DNA methyl NUC013 / 041 transferase inhibitor Attribute Current DNA methyl NUC013 / 041 transferase inhibitor Attribute Current DNA methyl NUC013 / 041 transferase inhibitor old y in solid tumors. - Increase CD8-T cells (cytotoxic T-lymphocytes, or CTL). Cytotoxic T cells expressing cell-surface CD8 are the most powerful effectors in the anticancer immune response and form the backbone of current successful cancer immunotherapies.37- Increase NK (natural killer cells). Natural killer cells are members of the innate immune system and promote cytotoxic activity against tumor independently from MHC recognition.38NK cell immunotherapy is safe and effective for patients with advanced stage leukemia.39- Increase NK granzyme B. Granzyme B and perforin proteins comprise the ammunition for NK cell-mediated killing.40- Increase NKT (natural killer T-cells). Have shown antitumor activity by linking innate and adaptive system.41NKT cells have the capacity to mount strong anti- tumor responses and have thus become a major focus in the development of effective cancer immunotherapy.42- Increase NKT granzyme B. The perforin / granzyme-B pathway of NKT cells caused up to 85% of cytotoxicity.43- Decrease Treg cells (T-regulatory cells). In a meta-analysis of all types of cancer, Treg had a negative impact on patient survival.44Treg cells lower the immune response to cancer cells by infiltrating into the tumor cells, favoring tumor progression and inhibiting the activation of T-cells to respond to cancer cells. Depletion of Treg cells can pave the way to an effective immunotherapy by activating the immune responses against cancer.45- Increase CD8 to Treg ratio. A low CD8 / Treg is associated with poor outcome in a number of cancers, including ovarian,46bladder cancer47and breast cancer.48- Increase CD4 to Treg ratio. The predictive value of CD4 / Treg is not as well accepted as that of CD8 / Treg, but has been established for colorectal cancer.49- Decrease MDSC (myeloid derived suppressor cells). MDSC represent the main immunosuppressive cells present in the tumor microenvironment that sustain cancer progression. MDSC are a heterogeneous group of immature myeloid cells with a potent activity against T-cells. Clinical studies have shown that MDSC levels in the peripheral blood of cancer patients correlates with tumor burden, stage and with poor prognosis in multiple malignancies. Thus, MDSC are the major obstacle to many cancer immunotherapies and their targeting may be a beneficial strategy.50- Decrease MAGE-A expression on tumor cells: Decitabine is widely reported in the literature to increase MAGE-A expression, and there is interest in combining decitabine with T-cell immunotherapy for this reason. NUC041 shows a decrease in MAGE expression, which is probably the result of CD8-T cells targeting MAGE- A expressing tumor cells. This finding has not been reported with any other DNMTI, probably because they are reported to decrease CD8-T cells mediated by Treg expansion.51This hypothesis is supported by the fact (1) 91.2% of tumor cells express MAGE-A at baseline and (2) that the proportion of decrease in MAGE-A positive cells in NUC041 treated tumors is 29.6%, while that of live tumor cells is almost identical, 31.1%. The current drugs that come closest to achieving these goals are DNMTIs, by derepressing genes that are repressed in cancer. They have been shown in a variety of publications to achieve some of these goals, namely: approved DNMTIs have shown the potential to affect the immune system through multiple mechanisms, including: upregulation of tumor antigen expression, increased MHC class I expression, and blunting of myeloid derived suppressor cells (MDSC) expansion.52However, they suffer from three important shortcomings: - While they are approved for the treatment of hematologic malignancies, they have shown limited ability to treat solid tumors in vivo, and particularly in patients.53,54These findings are concordant with data presented in table 5, where the vast majority of the studies were in hematologic tumors. - The bulk of the data demonstrate that current DNMTI, upregulate Treg cells or at best have no effect. Only one study has claimed that Treg cell proliferation is decreased by decitabine and CD8-T cells increased in patients with AML.55Treg are considered immunosuppressive and may decrease CD8-T proliferation.56- While approved DNMTI have been shown to increase expression of MAGE-A, there is no evidence that these drugs can effectively treat MAGE-A expressing cells without addition of another immunotherapy, such as a vaccine57or T-cell therapy.58NUC013 / 041 has been shown to achieve all of the above goals, and to have a better profile as an immunotherapeutic. In particular NUC013 and gemcitabine have been shown to be ribonucleotide reductase (RNR) inhibitors, and one of its subunits of RNR (RRM2, small subunit 2) has been shown to inhibit Treg. Overexpression of the RRM2 small subunit of RNR creates an immunosuppressive tumor-immune microenvironment. The infiltration of antitumor immune cells such as CD8-T was significantly lower in the RRM2-high group than in the RRM2-low group, whereas immunosuppressive Treg cells were more abundant in RRM2-high tumors.59High RRM2 levels were also correlated with the number of Treg cells in other studies.60,61Gemcitabine has been shown to decrease Treg cells in the blood of pancreatic cancer patients.62Suppression of Treg cells, results in increases in CD4 / Treg and CD8 / Treg, and the latter is considered one of the best markers of disease severity and progression.63,64While current DNMTI are only approved for hematologic malignancies, gemcitabine is only approved for the treatment of solid tumors.65As NU013 has the same base as 5- azacytidine or decitabine, and the sugar of gemcitabine, it is able to successfully treat both types of tumors.4,6Previously, NUC013 had been shown to be able to treat colon cancer and NUC041 non-small cell lung cancer, both in a mouse models. However, NUC041 had not been shown to be able to treat difficult to treat solid tumors with poor prognosis, which were not previously treatable with a DNMTI. NUC013 and NUC041 have shown statistically significant improvements in tumor growth suppression and, where possible, based on constraints of study duration, survival, in mouse xenografts of the following: Xenografts of renal cancer Two different cell lines were implanted in nude mice. The 786-O cell line is a human clear cell renal cell carcinoma (ccRCC) cell line that is Von Hippel Lindau (VHL) defective and p53 mutant. The Caki-1 cell line originates from metastatic ccRCC and is wild type (WT) VHL and p53.66,67As in Figure 4A, mice with 786-O had significantly lower (P < 0.05) tumor volume in treated (0.56 mg qod in FS-14) versus control animals on study days 4-42 (Figure 2A). In the treated group, one mouse was cured, with no tumor found on histopathology. No mortality was recorded in NUC041 treated or vehicle control. Mice with Caki-1 (Figure 4B) had significantly lower (P < 0.05) tumor volumes noted in treated (0.56 mg qod in FS-14) versus control animals on study days 10-35. The study was terminated as per protocol on study day 42, hence the median survival of treated mice versus control mice could not be determined (> 42 days (undefined) versus 42 days, p = 0.061) (Figure 4C). Three mice were euthanized for weight loss > 20% in the control group versus none in treated group. In Figure 4D, mice with Caki-1 had significantly lower tumor volumes noted in treated (0.8 mg qod in FS-19) versus control animals, (P < 0.05) study days 6-42. Mortality was insufficient for evaluation of survival (death of one mouse in NUC041 treated and vehicle control groups). A comparison was performed on effectiveness of 0.8 mg dose on study day 34 versus 0.56 mg dose on study day 35 (day 35 was the last day when all vehicle control mice in the 0.56 mg study were alive). Tumors were rank ordered by starting tumor volume, then a ratio calculated for matched pairs of tumor volume from treated animals divided by tumor volume in control animals. A comparison was then performed of these ratios for the two doses. The mean tumor ratio (NUC041 / vehicle) in the 0.56 mg study was 0.66 versus 0.36 in the 0.8 mg study (p = 0.044). Pancreatic xenograft models CFPAC-1 is a human ductal adenocarcinoma cell line and is p53 mutant.4SW1990 is a cell line derived from metastatic pancreatic adenocarcinoma and is variously described as p53 WT68or mutant.4Based on a tolerability study in immunocompetent CD-1 male mice (data not shown), mice implanted with CFPAC-1 were started on a dose of 1.2 mg SC qod of NUC041 in FS-19 or PBS (n =10 per group) (see Figure 5A). Because of toxicity, the dose was reduced to 1 mg SC qod on day 6 and then 0.8 mg SC qod on day 16. Two mice died because of drug toxicity in the treated group (one day 4, the other day 6). Tumor volume in treated mice was significantly smaller than control mice on study days 2-42 (p < 0.0001). There was not sufficient mortality to determine survival. Tumor volumes in treated mice implanted with SW1990 were significantly smaller than control mice on study days 4 through 36 (p < 0.025) (see Figure 5B). The median survival of treated mice was significantly longer than that of control mice (42 versus 30 days, p < 0.0001) (see Figure 4C). The DU4475 cell line is derived from a Triple-Negative Breast Cancer TNBC and is reported to be p53 wild type.4Treated mice implanted with DU4475 were initially started on two different doses (see Figure 6A) but mice, particularly in the 0.8 mg group, required dose adjustments, hence the dose was decreased in both treated groups to 0.5 mg qod on study day 8 because of concern that weight loss in the control group could complicate assessment of drug toxicity and efficacy. Tumor volume in treated mice was significantly smaller than that of control mice: In the 0.8 mg group, on study days 4-20 (p < 0.05) and in the 0.6 mg group, on study days 4-20 (p < 0.01). The median survival of 0.8 mg treated mice versus control mice was 21 versus 18 days (p = 0.019) and for the 0.6 mg treated mice 25 to 18 days (p = 0.0032) (see Figure 6B). The xenograft mouse models demonstrate the efficacy of NUC041 with significant inhibition of tumor growth and further improvements with the introduction of the FS-19 formulation, leading to decreased vehicle toxicity and improved pharmacokinetics. Significant inhibition of tumor growth was demonstrated in models of renal cancer (786-O and Caki-1), pancreatic cancer (CFPAC-1 and SW1990) and triple negative breast cancer (DU4475). Improvements were shown in survival, where there were a sufficient number of events in the 42-day study period, in all three tissue types. Renal Cell Carcinoma (RCC) is classified as an immunogenic tumor, like melanoma, because of the occurrence of spontaneous regression, response to immunotherapy and high level of T-cell infiltration.69Immune checkpoint inhibitors are becoming the first-line treatment of metastatic RCC.70However, high-dose interleukin-2 (IL-2) is the only systemic therapy that has been associated with inducing durable complete remissions, though only in about 5% of patients.71IL-2 activates lymphokine activated killer (LAK) cells leading to lysis of tumor cells. LAK cells are comprised mainly of NK and T-cells.72However, administration of high-dose IL-2 requires admission to a hospital because patients can suffer severe side effects from treatment.73In our study, one mouse implanted with 786-O and treated with NUC041 was cured, with no tumor found on histopathology. Decitabine has been reported to have no activity in a mouse xenograft of 786-O (VHL-defective).74Since both NUC013 and decitabine are reported to caused p53 independent apoptosis at submicromolar doses,1these results suggest that the difference in activity against 786-O may be the antitumor immune response induced by NUC041. Pancreatic cancer remains a cancer with a very poor prognosis and gemcitabine, a ribonucleotide reductase inhibitor, remains a mainstay of treatment.75The main cure for pancreatic cancer is surgical resection and all other treatment modalities provide low survival rates. Currently, many clinical trials seek to assess the efficacy of immunotherapeutic strategies, but none of these studies have shown dramatic results.76In our study, animals with pancreatic cancer demonstrated a good response to treatment, particularly animals implanted with CFPAC-1, when compared to a report in the literature of treatment with gemcitabine of mice with the same starting tumor volume.77Breast cancer is categorized into three major subtypes based on the presence or absence of molecular markers for estrogen (ER) or progesterone receptors (PR) and human epidermal growth factor 2 (ERBB2, formerly HER2): Triple-Negative Breast Cancer (TNBC) lacks all three standard molecular markers (15% of patients).78Most new treatment options for metastatic breast cancer recently approved by the Food and Drug Administration (FDA) are only effective for ER / PR (+) or ERBB2 (+) metastatic tumors, and the subset of patients with metastatic TNBC are treated with chemotherapy alone. Hence there is a need for novel therapies and immunotherapy may be a viable treatment strategy.79DU4475 is a model of an aggressive triple negative cancer and despite issues related to optimal dosing, NUC041 has demonstrated significant inhibition of tumor growth and improved survival. As discussed above, while NUC041 is more effective than decitabine at treating RCC, there are no reports in the literature of treatment of TNBC or pancreatic cancer by a DNMTI. Histopathology of renal cell cancer Caki-1 tumors and quantitation of tumor necrosis Tumors from mice inoculated with Caki-1 were fixed in 10% neutral buffered formalin. The fixed neoplasm was trimmed, processed, embedded and microtomed (approximately 4 µm sections). Tissue sections were mounted on glass slides, stained with hematoxylin and eosin (H&E), and coverslipped. Slides were submitted to a veterinary pathologist for a histopathologic evaluation. HALO software was trained to classify regions of the tissue into active tumor and non-tumor components. Non tumor components included stroma and areas of tumor necrosis, and from these data the percentage of tumor necrosis was determined. Mice with Caki-1 implants had significantly lower (p < 0.05) tumor volumes in treated (0.56 mg qod in FS-14) compared to control animals on days 10-35. The study was terminated as per the protocol on day 42; hence, the median survival of treated mice vs. control mice could not be determined. Three mice were euthanized for > 20% weight loss in the control group and none in the treated group. These two groups of mice underwent a histologic study to quantitate the amount of tumor necrosis. Necrosis was present in 22.0% of untreated tumor cells (Figures 7A and 7B) vs 9.4% of treated tumor cells (Figures 8A and B), p = 0.021 (see Table 6). Total Area Tissue Area Necrosis Necrosis Average Total Area Tissue Area Necrosis Average Mouse ID Area Necrosis necrosis δmm2ε δmm2ε2δ%ε % ith Caki-1 at end of study. Mice were identified through a numbering system. Mice1-1 through 2-5 were treated with vehicle (PBS), while mice 3-1 through 4-5 were treated with 0.56 mg NUC041 SC in FS-14 qod for up to 42 days. Because of their low mortality, Caki-1 inoculated mice were selected for a substudy to quantitate the amount of necrosis in the tumors. Necrosis was of interest because a study with mice inoculated with NCI-H460 had demonstrated on histology a marked inflammatory response, necrosis and in some cases ulceration.6A significant decrease in necrosis was observed in the treated vs. the control group. Tumor necrosis is associated with rapid tumor growth and is a predictor of poor prognosis in cancer.80,81These results demonstrate that NUC041 decreases the proportion of necrotic cells in tumors of treated animals. Similar experiments could be performed with other pyrimidine analogs, such as other variants of NUC013, and achieve the same results. These experiments could be performed in dogs and non-human primates. These experiments could be performed with a lower percentage of DSPE-PEG2000-NH2. These experiments could be performed with a higher percentage of DSPE-PEG2000-NH2.These experiments could be performed with other PEG formulations. Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application. Accordingly, it is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Any publication, document, patent, patent application or internet content referred to herein should be construed as incorporated by reference each in their entirety for all purposes. REFERENCES 1. Feinberg AP. The Key Role of Epigenetics in Human Disease Prevention and Mitigation. N Engl J Med.2018 Apr 5;378(14):1323-1334. 2. Daifuku R. Pharmacoepigenetics of novel nucleoside DNA methyltransferase inhibitors. In: Pharmacoepigenetics. Cacabellos R (Ed.). Elsevier.2019, (10):425-435. 3. Daifuku R, Gall A, Sergueev DS. Fluorinated pyrimidine analogs and methods of us thereof. US 9,493,500 B2 Nov 15, 2016. 4. Daifuku R, Hu Z, Saunthararajah Y. 5-aza-2',2'-Difluoro Deoxycytidine (NUC013): A Novel Nucleoside DNA Methyl Transferase Inhibitor and Ribonucleotide Reductase Inhibitor for the Treatment of Cancer. Pharmaceuticals (Basel). 2017 Jul 20;10(3):65. 5. Daifuku R, Sergueev DS. Silylated pyrimidine prodrugs and methods of their use. US 10,479,807 B2 Nov 19, 2019. 6. Daifuku R, Grimes S, Stackhouse M. NUC041, a Prodrug of the DNA Methytransferase Inhibitor 5-aza-2',2'-Difluorodeoxycytidine (NUC013), Leads to Tumor Regression in a Model of Non-Small Cell Lung Cancer. Pharmaceuticals (Basel). 2018 Apr 23;11(2):36. 7. Daifuku R, Grimes S, Stackhouse M. NUC041, a Prodrug of the DNA Methytransferase Inhibitor 5-aza-2',2'-Difluorodeoxycytidine (NUC013), Leads to Tumor Regression in a Model of Non-Small Cell Lung Cancer. Pharmaceuticals (Basel). 2018 Apr 23;11(2):36. 8. Sharpe M, Easthope SE, Keating GM, Lamb HM. Polyethylene glycol-liposomal doxorubicin: a review of its use in the management of solid and haematological malignancies and AIDS-related Kaposi's sarcoma. Drugs. 2002;62(14):2089-126. 9. Prajna Mishra, Bismita Nayak, R.K. Dey. PEGylation in anti-cancer therapy: An overview. Asian Journal of Pharmaceutical Sciences. 2016. 11(3):337-348. 10. Shi D, Beasock D, Fessler A, Szebeni J, Ljubimova JY, Afonin KA, Dobrovolskaia MA. To PEGylate or not to PEGylate: Immunological properties of nanomedicine's most popular component, polyethylene glycol and its alternatives. Adv Drug Deliv Rev. 2022 Jan;180:114079. 11. Dong Y, Wang J, Tao QS, Shen YY, Zhai ZM. [Effectiveness and Mechanism of Decitabine Maintenance Therapy in Patients with Medium and Low-risk Acute Myeloid Leukemia]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct;30(5):1369-1375. Chinese. 12. Luker AJ, Graham LJ, Smith TM Jr, Camarena C, Zellner MP, Gilmer JS, Damle SR, Conrad DH, Bear HD, Martin RK. The DNA methyltransferase inhibitor, guadecitabine, targets tumor-induced myelopoiesis and recovers T cell activity to slow tumor growth in combination with adoptive immunotherapy in a mouse model of breast cancer. BMC Immunol. 2020 Feb 27;21(1):8. 13. Stübig T, Badbaran A, Luetkens T, Hildebrandt Y, Atanackovic D, Binder TM, Fehse B, Kröger N. 5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity. Mediators Inflamm.2014;2014:418292. 14. Gang, A., Frøsig, T., Brimnes, M. et al.5-Azacytidine treatment sensitizes tumor cells to T-cell mediated cytotoxicity and modulates NK cells in patients with myeloid malignancies. Blood Cancer Journal 4, e197 (2014). 4. 15. Gao XN, Lin J, Wang LL, Yu L. Demethylating treatment suppresses natural killer cell cytolytic activity. Mol Immunol. 2009 Jun;46(10):2064-70.. 16. Cany J, Roeven MWH, Hoogstad-van Evert JS, Hobo W, Maas F, Franco Fernandez R, Blijlevens NMA, van der Velden WJ, Huls G, Jansen JH, Schaap NPM, Dolstra H. Decitabine enhances targeting of AML cells by CD34+progenitor-derived NK cells in NOD / SCID / IL2Rgnullmice. Blood. 2018 Jan 11;131(2):202-214. doi: 10.1182 / blood-2017-06-790204. 17. Sutherland MK, Yu C, Anderson M, Zeng W, van Rooijen N, Sievers EL, Grewal IS, Law CL. 5-azacytidine enhances the anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia. MAbs. 2010 Jul-Aug;2(4):440-8. 18. Cany J, Roeven MWH, Hoogstad-van Evert JS, Hobo W, Maas F, Franco Fernandez R, Blijlevens NMA, van der Velden WJ, Huls G, Jansen JH, Schaap NPM, Dolstra H. Decitabine enhances targeting of AML cells by CD34+progenitor-derived NK cells in NOD / SCID / IL2Rgnullmice. Blood. 2018 Jan 11;131(2):202-214. 19. Li X, Zhang M, Cai S, Wu Y, You Y, Wang X, Wang L. Concentration-Dependent Decitabine Effects on Primary NK Cells Viability, Phenotype, and Function in the Absence of Obvious NK Cells Proliferation-Original Article. Front Pharmacol. 2021 Oct 25;12:755662. 20. Dong Y, Wang J, Tao QS, Shen YY, Zhai ZM. [Effectiveness and Mechanism of Decitabine Maintenance Therapy in Patients with Medium and Low-risk Acute Myeloid Leukemia]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct;30(5):1369-1375. 21. Gang, A., Frøsig, T., Brimnes, M. et al.5-Azacytidine treatment sensitizes tumor cells to T-cell mediated cytotoxicity and modulates NK cells in patients with myeloid malignancies. Blood Cancer Journal 4, e197 (2014). 4. 22. Zhigarev D, Varshavsky A, MacFarlane AW 4th, Jayaguru P, Barreyro L, Khoreva M, Dulaimi E, Nejati R, Drenberg C, Campbell KS. Lymphocyte Exhaustion in AML Patients and Impacts of HMA / Venetoclax or Intensive Chemotherapy on Their Biology. Cancers (Basel). 2022 Jul 10;14(14):3352. 23. Dong Y, Wang J, Tao QS, Shen YY, Zhai ZM. [Effectiveness and Mechanism of Decitabine Maintenance Therapy in Patients with Medium and Low-risk Acute Myeloid Leukemia]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct;30(5):1369-1375. Chinese. 24. Landman, S., van der Horst, C., van Erp, P.E.J. et al. Immune responses to azacytidine in animal models of inflammatory disorders: a systematic review. J Transl Med 19, 11 (2021). 2. 25. Amaro A, Reggiani F, Fenoglio D, et al. Landman S, van der Horst C, van Erp PEJ, Joosten I, de Vries R, Koenen HJPM. Immune responses to azacytidine in animal models of inflammatory disorders: a systematic review. J Transl Med.2021 Jan 6;19(1):11. 26. Adriana Amaro, Francesco Reggiani, Daniela Fenoglio et al. Guadecitabine increases response to combined anti-CTLA-4 and anti-PD-1 treatment in mouse melanoma in vivo by controlling the responses of T-cells, myeloid derived suppressor cells and NK cells, 03 January 2023, PREPRINT (Version 1) available at Research Square [https: / / doi.org / 10.21203 / rs.3.rs-2408433 / v1] 27. Fang Y, Yuan XD, Liu HH, Xiang L, Chen LM, Fan YC, Gao S, Wang K. 5-Aza-2'- deoxycytidine may enhance the frequency of T regulatory cells from CD4+naïve T cells isolated from the peripheral blood of patients with chronic HBV infection. Expert Rev Clin Immunol. 2021 Feb;17(2):177-185. doi: 10.1080 / 1744666X.2020.1866987. Epub 2021 Jan 13. PMID: 33353450. 28. Tsirogianni M, Grigoriou E, Kapsimalli V, Dagla K, Stamouli M, Gkirkas K, Konsta E, Karagiannidou A, Gkodopoulos K, Stavroulaki G, Pappa V, Angelopoulou M, Lowdell M, Tsirigotis P. Natural killer cell cytotoxicity is a predictor of outcome for patients with high risk myelodysplastic syndrome and oligoblastic acute myeloid leukemia treated with azacytidine. Leuk Lymphoma. 2019 Oct;60(10):2457-2463. 29. Luker AJ, Graham LJ, Smith TM Jr, Camarena C, Zellner MP, Gilmer JS, Damle SR, Conrad DH, Bear HD, Martin RK. The DNA methyltransferase inhibitor, guadecitabine, targets tumor-induced myelopoiesis and recovers T cell activity to slow tumor growth in combination with adoptive immunotherapy in a mouse model of breast cancer. BMC Immunol. 2020 Feb 27;21(1):8. 30. Tsirogianni M, Grigoriou E, Kapsimalli V, Dagla K, Stamouli M, Gkirkas K, Konsta E, Karagiannidou A, Gkodopoulos K, Stavroulaki G, Pappa V, Angelopoulou M, Lowdell M, Tsirigotis P. Natural killer cell cytotoxicity is a predictor of outcome for patients with high risk myelodysplastic syndrome and oligoblastic acute myeloid leukemia treated with azacytidine. Leuk Lymphoma. 2019 Oct;60(10):2457-2463. 31. Zhou J, Yao Y, Shen Q, Li G, Hu L, Zhang X. Demethylating agent decitabine disrupts tumor-induced immune tolerance by depleting myeloid-derived suppressor cells. J Cancer Res Clin Oncol.2017 Aug;143(8):1371-1380. 32. Li W, Sang M, Hao X, Wu Y, Shan B. CTLA-4 blockade combined with 5-aza-2'-deoxycytidine enhances the killing effect of MAGE-A family common antigen peptide-specific cytotoxic T cells on breast cancer. Oncol Rep.2020 Oct;44(4):1758-1770. 33. Bert T, Lubomierski N, Gangsauge S, Münch K, Printz H, Prasnikar N et al. Expression spectrum and methylation-dependent regulation of melanoma antigen- encoding gene family members in pancreatic cancer cells. Pancreatology.2002;2(2):146- 54. 34. Kim KH, Choi JS, Kim IJ, Ku JL, Park JG. Promoter hypomethylation and reactivation of MAGE-A1 and MAGE-A3 genes in colorectal cancer cell lines and cancer tissues. World J Gastroenterol. 2006 Sep 21;12(35):5651-7. 35. Krishnadas DK, Shusterman S, Bai F, Diller L, Sullivan JE, Cheerva AC, George RE, Lucas KG. A phase I trial combining decitabine / dendritic cell vaccine targeting MAGE- A1, MAGE-A3 and NY-ESO-1 for children with relapsed or therapy-refractory neuroblastoma and sarcoma. Cancer Immunol Immunother. 2015 Oct;64(10):1251-60. 36. Shi X, Chen X, Fang B, Ping Y, Qin G, Yue D, Li F, Yang S, Zhang Y. Decitabine enhances tumor recognition by T cells through upregulating the MAGE-A3 expression in esophageal carcinoma. Biomed Pharmacother. 2019 Apr;112:108632. 37. Raskov H, Orhan A, Christensen JP, Gögenur I. Cytotoxic CD8+T cells in cancer and cancer immunotherapy. Br J Cancer. 2021 Jan;124(2):359-367. 38. Ghazvinian Z, Abdolahi S, Tokhanbigli S, Tarzemani S, Piccin A, Reza Zali M, Verdi J, Baghaei K. Contribution of natural killer cells in innate immunity against colorectal cancer. Front Oncol.2023 Jan 4;12:1077053. 39. Myers, J.A., Miller, J.S. Exploring the NK cell platform for cancer immunotherapy. Nat Rev Clin Oncol 18, 85–100 (2021). 40. Leong JW, Fehniger TA. Human NK cells: SET to kill. Blood. 2011 Feb 24;117(8):2297-8. 41. Bae EA, Seo H, Kim IK, Jeon I, Kang CY. Roles of NKT cells in cancer immunotherapy. Arch Pharm Res.2019 Jul;42(7):543-548. 42. Webb TJ, Yuan W, Meyer E, Dellabona P. Editorial: NKT Cells in Cancer Immunotherapy. Front Immunol.2020 Jun 23;11:1314. 43. Metelitsa LS, Weinberg KI, Emanuel PD, Seeger RC. Expression of CD1d by myelomonocytic leukemias provides a target for cytotoxic NKT cells. Leukemia. 2003 Jun;17(6):1068-77. 44. Shang, B., Liu, Y., Jiang, Sj. et al. Prognostic value of tumor-infiltrating FoxP3+regulatory T cells in cancers: a systematic review and meta-analysis. Sci Rep 5, 15179 (2015). 45. Zeng G, Jin L, Ying Q, Chen H, Thembinkosi MC, Yang C, Zhao J, Ji H, Lin S, Peng R, Zhang M, Sun D. Regulatory T Cells in Cancer Immunotherapy: Basic Research Outcomes and Clinical Directions. Cancer Manag Res. 2020 Oct 21;12:10411-10421. 46. Preston CC, Maurer MJ, Oberg AL, Visscher DW, Kalli KR, Hartmann LC, Goode EL, Knutson KL. The ratios of CD8+ T cells to CD4+CD25+ FOXP3+ and FOXP3- T cells correlate with poor clinical outcome in human serous ovarian cancer. PLoS One. 2013 Nov 14;8(11):e80063. 47. Baras AS, Drake C, Liu JJ, Gandhi N, Kates M, Hoque MO, Meeker A, Hahn N, Taube JM, Schoenberg MP, Netto G, Bivalacqua TJ. The ratio of CD8 to Treg tumor- infiltrating lymphocytes is associated with response to cisplatin-based neoadjuvant chemotherapy in patients with muscle invasive urothelial carcinoma of the bladder. Oncoimmunology.2016 Feb 18;5(5):e1134412. 48. Goda, N., Sasada, S., Shigematsu, H. et al. The ratio of CD8 + lymphocytes to tumor-infiltrating suppressive FOXP3 + effector regulatory T cells is associated with treatment response in invasive breast cancer. Discov Onc 13, 27 (2022).5 49. Xu T, Lu J, An H. The relative change in regulatory T cells / T helper lymphocytes ratio as parameter for prediction of therapy efficacy in metastatic colorectal cancer patients. Oncotarget. 2017 Nov 21;8(65):109079-109093. 50. De Cicco P, Ercolano G, Ianaro A. The New Era of Cancer Immunotherapy: Targeting Myeloid-Derived Suppressor Cells to Overcome Immune Evasion. Front Immunol.2020 Jul 30;11:1680. 51. Stübig T, Badbaran A, Luetkens T, Hildebrandt Y, Atanackovic D, Binder TM, Fehse B, Kröger N. 5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity. Mediators Inflamm.2014;2014:418292. 52. Terracina KP, Graham LJ, Payne KK, Manjili MH, Baek A, Damle SR, Bear HD. DNA methyltransferase inhibition increases efficacy of adoptive cellular immunotherapy of murine breast cancer. Cancer Immunol Immunother.2016 Sep;65(9):1061-73. 53. Linnekamp JF, Butter R, Spijker R, Medema JP, van Laarhoven HWM. Clinical and biological effects of demethylating agents on solid tumours - A systematic review. Cancer Treat Rev.2017 Mar;54:10-23. 53. Gemcitabine Prescribing Information https: / / www.drugs.com / pro / gemcitabine.html#s-34090-1 54. Nervi C, De Marinis E, Codacci-Pisanelli G. Epigenetic treatment of solid tumours: a review of clinical trials. Clin Epigenetics. 2015 Dec 10;7:127. 55. Dong Y, Wang J, Tao QS, Shen YY, Zhai ZM. [Effectiveness and Mechanism of Decitabine Maintenance Therapy in Patients with Medium and Low-risk Acute Myeloid Leukemia]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct;30(5):1369-1375. Chinese. 56. Stübig T, Badbaran A, Luetkens T, Hildebrandt Y, Atanackovic D, Binder TM, Fehse B, Kröger N. 5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity. Mediators Inflamm.2014;2014:418292. 57. Krishnadas DK, Shusterman S, Bai F, Diller L, Sullivan JE, Cheerva AC, George RE, Lucas KG. A phase I trial combining decitabine / dendritic cell vaccine targeting MAGE- A1, MAGE-A3 and NY-ESO-1 for children with relapsed or therapy-refractory neuroblastoma and sarcoma. Cancer Immunol Immunother. 2015 Oct;64(10):1251-60. 58. Shi X, Chen X, Fang B, Ping Y, Qin G, Yue D, Li F, Yang S, Zhang Y. Decitabine enhances tumor recognition by T cells through upregulating the MAGE-A3 expression in esophageal carcinoma. Biomed Pharmacother. 2019 Apr;112:108632. 59. Mazzu YZ, Armenia J, Nandakumar S, Chakraborty G, Yoshikawa Y, Jehane LE, Lee GM, Atiq M, Khan N, Schultz N, Kantoff PW. Ribonucleotide reductase small subunit M2 is a master driver of aggressive prostate cancer. Mol Oncol. 2020 Aug;14(8):1881- 1897. 60. Xiong W, Zhang B, Yu H, Zhu L, Yi L, Jin X. RRM2 Regulates Sensitivity to Sunitinib and PD-1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway. Adv Sci (Weinh). 2021 Sep;8(18):e2100881. 61. Li Y, Fu W, Geng Z, Song Y, Yang X, He T, Wu J, Wang B. A pan-cancer analysis of the oncogenic role of ribonucleotide reductase subunit M2 in human tumors. PeerJ. 2022 Nov 28;10:e14432. 62. Eriksson E, Wenthe J, Irenaeus S, Loskog A, Ullenhag G. Gemcitabine reduces MDSCs, tregs and TGFβ-1 while restoring the teff / treg ratio in patients with pancreatic cancer. J Transl Med. 2016 Sep 29;14(1):282. 63. Xu T, Lu J, An H. The relative change in regulatory T cells / T helper lymphocytes ratio as parameter for prediction of therapy efficacy in metastatic colorectal cancer patients. Oncotarget. 2017 Nov 21;8(65):109079-109093. 64. Preston CC, Maurer MJ, Oberg AL, Visscher DW, Kalli KR, Hartmann LC, Goode EL, Knutson KL. The ratios of CD8+ T cells to CD4+CD25+ FOXP3+ and FOXP3- T cells correlate with poor clinical outcome in human serous ovarian cancer. PLoS One. 2013 Nov 14;8(11):e80063. 65. https: / / www.cancer.gov / about-cancer / treatment / drugs / gemcitabinehydrochloride 66. https: / / tp53.isb-cgc.org / view_data?bq_view_name=CellLineDownload 67. Brodaczewska KK, Szczylik C, Fiedorowicz M, Porta C, Czarnecka AM. Choosing the right cell line for renal cell cancer research. Mol Cancer.2016 Dec 19;15(1):83. 68. https: / / www.atcc.org / products / cells_and_microorganisms / cell_lines / human / tumor _cell_panels / panels_by_tissue_type / pancreatic_cancer_cell_panel / tcp-2060. 69. Itsumi M, Tatsugami K. Immunotherapy for renal cell carcinoma. Clin Dev Immunol. 2010;2010:284581. 70. Angulo JC, Shapiro O. The Changing Therapeutic Landscape of Metastatic Renal Cancer. Cancers (Basel). 2019 Aug 22;11(9):1227. 71. PDQ Adult Treatment Editorial Board. Renal cell cancer treatment (PDQ®): Health professional version. https: / / www.ncbi.nlm.nih.gov / books / NBK65815 / #CDR0000062894__1. Accessed 15 Sep2019. 72. Wendel P, Reindl LM, Bexte T, Künnemeyer L, Särchen V, Albinger N et al. Arming Immune Cells for Battle: A Brief Journey through the Advancements of T and NK Cell Immunotherapy. Cancers (Basel). 2021 Mar 23;13(6):1481. 73. Amin A, White RL. Interleukin-2 in Renal Cell Carcinoma: A Has-Been or a Still- Viable Option? J Kidney Cancer VHL. 2014 Nov 23;1(7):74-83. 74. Alleman WG, Tabios RL, Chandramouli GV, Aprelikova ON, Torres-Cabala C, Mendoza A et al. The in vitro and in vivo effects of re-expressing methylated von Hippel- Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2'-deoxycytidine. Clin Cancer Res.2004 Oct 15;10(20):7011-21. 75. Huang JH, Guo W, Liu Z. Discussion on gemcitabine combined with targeted drugs in the treatment of pancreatic cancer. World J Gastroenterol.2023 Jan 21;29(3):579-581. 76. Schizas D, Charalampakis N, Kole C, Economopoulou P, Koustas E, Gkotsis E et al. Immunotherapy for pancreatic cancer: A 2020 update. Cancer Treat Rev. 2020 Jun;86:102016. 77. Li Z, Zou X, Zhu H, Chen M, Zhao Y. Inhibitory effect of baicalein combined with gemcitabine in human pancreatic cancer cell lines. Oncol Lett.2018 Apr;15(4):5459-5464. 78. Waks AG, Winer EP. Breast Cancer Treatment: A Review. JAMA. 2019 Jan 22;321(3):288-300. 79. Emens LA. Immunotherapy in Triple-Negative Breast Cancer. Cancer J.2021 Jan- Feb 01;27(1):59-66. 80. Yee PP, Li W. Tumor necrosis: A synergistic consequence of metabolic stress and inflammation. Bioessays. 2021 Jul;43(7):e2100029. 81. Liu ZG, Jiao D. Necroptosis, tumor necrosis and tumorigenesis. Cell Stress. 2019 Dec 19;4(1):1-8.! !

Claims

CLAIMS 1. Formulation of a pyrimidine prodrug in a formulation comprising PEG, wherein the pyrimidine is 5-aza-2',2'-difluorodeoxycytidine (NUC013).

2. The formulation of claim 1, further comprising a phospholipid.

3. The formulation of claim 1 or 2, wherein PEG is DSPE-PEG2000-CN or DSPE- PEG2000-NH2.

4. The formulation of any one of claims 1 to 3, wherein PEG is at a concentration from greater than zero to 20% weight to weight.

5. The formulation of any one of claims 1 to 4, wherein the pyrimidine prodrug is 2',2'-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041) having the structure: .

6. Method of treatment of cancer with the formulation of any one of claims 1-5.

7. Use of the formulation of any one of claims 1-5 for treating cancer.

8. Use of the formulation of any one of claims 1-5 for the manufacture of a medicament for treating cancer.

9. Use of the formulation of any one of claims 1-5 for affecting both arms of the anti- tumor immune system.

10. Use of the formulation of any one of claims 1-5 for the manufacture of a medicament for affecting both arms of the anti-tumor immune system.

11. A formulation as defined in any one of claims 1-5 for treating cancer.

12. A formulation as defined in any one of claims 1-5 for affecting both arms of the anti-tumor immune system.

13. A method of treating cancer by stimulating both arms, innate and adaptive, of the immune system with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

14. A method of treating cancer by stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC, with 5-aza-2',2'- difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

15. A method of treating cancer by increasing the proportion of NK and NKT cells positive for granzyme B, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

16. A method of treating cancer by increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

17. A method of treating cancer leading to a decrease in the proportion of necrotic cells in the tumor, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

18. A method of treating cancer by decreasing the proportion of cells expressing MAGE-A, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

19. A method of treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer, with 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug.

20. The method of any one of claims 13 to 19, wherein the pyrimidine prodrug is 2',2'- difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041).

21. The method of any one of claims 13 to 19, wherein the formulation of the pyrimidine prodrug is a formulation comprising PEG.

22. The method of claim 21, wherein the formulation further comprises a phospholipid.

23. The method of claim 21 or 22, wherein PEG is DSPE-PEG2000-CN or DSPE- PEG2000-NH2.

24. The method of any one of claims 21 to 23, wherein PEG is at a concentration from greater than zero to 20% weight to weight.

25. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for stimulating CD8 T cells (cytotoxic T- lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC.

26. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for increasing the proportion of NK and NKT cells positive for granzyme B.

27. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells.

28. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for leading to a decrease in the proportion of necrotic cells in the tumor.

29. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for decreasing the proportion of cells expressing MAGE-A.

30. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer.

31. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC.

32. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for increasing the proportion of NK and NKT cells positive for granzyme B.

33. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells.

34. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for leading to a decrease in the proportion of necrotic cells in the tumor.

35. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicament for decreasing the proportion of cells expressing MAGE-A.

36. Use of 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for the manufacture of a medicamentfor treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer.

37. The use of any one of claims 25 to 36, wherein the pyrimidine prodrug is 2',2'- difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041).

38. The use of any one of claims 25 to 37, wherein the formulation of the pyrimidine prodrug is a formulation comprising PEG.

39. The use of claim 38, wherein the formulation further comprises a phospholipid.

40. The use of claim 38 or 39, wherein PEG is DSPE-PEG2000-CN or DSPE- PEG2000-NH2.

41. The use of any one of claims 38 to 40, wherein PEG is at a concentration from greater than zero to 20% weight to weight. 42.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by stimulating both arms, innate and adaptive, of the immune system. 43.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by stimulating CD8 T cells (cytotoxic T-lymphocytes), NK cells and NKT cells, while inhibiting Treg cells and MDSC. 44.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by increasing the proportion of NK and NKT cells positive for granzyme B. 45.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by increasing the ratio of CD8 T cytotoxic cells to Treg cells and CD4 T effector cells to Treg cells. 46.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer leading to a decrease in the proportion of necrotic cells in the tumor. 47.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating cancer by decreasing the proportion of cells expressing MAGE-A. 48.5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use in treating difficult to treat solid tumors, such as pancreatic cancer, renal cancer and triple negative breast cancer.

49. The 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use of any one of claims 37 to 43, wherein the pyrimidine prodrug is 2',2'-difluoro-5-azadeoxycytidine-trimethylsilyl (NUC041).

50. The 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use of any one of claims 37 to 44, wherein the formulation of the pyrimidine prodrug is a formulation comprising PEG.

51. The 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use of claim 50, wherein the formulation further comprises a phospholipid.

52. The 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use of claim 50 or 51, wherein PEG is DSPE-PEG2000-CN or DSPE-PEG2000-NH2.

53. The 5-aza-2',2'-difluorodeoxycytidine (NUC013), a pyrimidine prodrug thereof or a formulation of said pyrimidine prodrug for use of any one of claims 50 to 52, wherein PEG is at a concentration from greater than zero to 20% weight to weight. !

Images