Combinations of heteroarotinoids and glycolytic inhibitors for use as cancer treatments

Abstract

Drug combinations of at least one heteroarotinoid and at least one glycolytic inhibitor, which are effective as therapeutic (e.g., anti-cancer treatments), are disclosed. Also disclosed are pharmaceutical compositions and kits containing same, as well as methods of using such drug combinations. Non-limiting examples of heteroarotinoids include SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, NHet90, as well as any combinations thereof. Non-limiting examples of glycolytic inhibitors include 2-deoxy-D-glucose (2-DG); 2-fluoro-2-deoxy-D-glucose (2-FG); 2-chloro-2-deoxy-Dglucose (2-CG); 2-bromo-2-deoxy-D-glucose (2-BG); acetylated derivatives of 2-DG; 3- bromopyruvate (3-BP); 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6- aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol; and metformin (MET).

Description

Electronically Transmitted: August 20, 2024 OU 2023-039 PCT COMBINATIONS OF HETEROAROTINOIDS AND GLYCOLYTIC INHIBITORS FOR USE AS CANCER TREATMENTS CROSS REFERENCE TO RELATED APPLICATIONS / INCORPORATION BY REFERENCE STATEMENT

[0001] This application claims benefit of U.S. Provisional Application Ser. No.63 / 578,180 filed August 23, 2023. The entire contents of the above-referenced patent application(s) are hereby expressly incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant No. 1R01CA200126 awarded by the National Cancer Institute of the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0003] Despite advanced screening programs and HPV vaccines, cervical cancer remains the fourth deadliest cancer in women worldwide. Although patients with early-stage cervical cancer have better outcomes with existing standards of care (chemoradiation or a combination of treatment modalities), these are highly toxic and often negatively impact the quality of life. Moreover, those with advanced and recurrent diseases have complicated clinical management and are often incurable. Thus, there is an unmet need to develop effective therapeutic alternatives focusing on new investigational agents and drug combinations for cervical cancer, as well as other cancers, which will widen this therapeutic window either by improving efficacy, lowering treatment-related toxicity, or, ideally, both. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 shows that SHetA2 targets mitochondrial respiration (O2 consumption rate) in cervical cancer cells: C-33A (A), Ca Ski (B) and SiHa (C). Cervical cancer cells were treated with 10 µM SHetA2 for 4 hours. OCR was measured using the Seahorse XFe96 analyzer and  cell mito-stress assay. Data are shown as mean±SD. **** p ≤ 0.0001 when compared with respective control.

[0005] FIG.2 shows that SHetA2 targets mitochondrial respiration in three cervical cancer cell lines: C-33A (lefthand bars), Ca Ski (center bars), SiHa (righthand bars). (A) SHetA2 stops mitochondrial basal respiration (OCR, measured at starting point before injection of any substance representing the basic cellular ATP demand), (B) SHetA2 stops ATP production (OCR measured after the injection of the ATP synthase inhibitor oligomycin representing ATP produced by the mitochondria that contributes to meeting the energetic needs of the cell). (C) SHetA2 targets Maximal Respiration (OCR measured after adding FCCP / uncoupler). Data are shown as mean±SD. **** p ≤ 0.0001 when compared with respective control.

[0006] FIG.3 shows that SHetA2 targets mitochondrial respiration in three cervical cancer cell lines: C-33A (lefthand bars), Ca Ski (center bars), SiHa (righthand bars). (A) SHetA2 targets Spare respiratory capacity. (B) SHetA2 targets Proton leak (Remaining basal OCR not coupled to ATP production after addition of antimycin A and rotenone). Data are shown as mean±SD. **** p ≤ 0.0001 when compared with respective control.

[0007] FIG.4 shows that cells of cervical cancer cell lines C-33A (A), Ca Ski (B) and SiHa (C) respond to SHetA2 by increasing the glycolysis. Cervical cancer cells were treated with SHetA2 for 4 hours and ECAR was measured using the Seahorse XF-96 analyzer and glycolytic rate assay. Data are shown as mean±SD and analyzed with a t-test. ** p ≤ 0.01, **** p ≤ 0.0001 when compared with respective controls.

[0008] FIG. 5 shows that cells of cervical cancer cell lines C-33A (lefthand bars), Ca Ski (center bars) and SiHa (righthand bars) respond to SHetA2 by increasing the glycolysis. (A) Basal PER, representing (total protons exported by cells measured represent the basic cellular ATP demand), (B) Basal glycol PER (PER derived from glycolysis), and (C) extra compensatory glycolysis (the rate of glycolysis in cells following the addition of Rotenone and Antimycin A to effectively inhibit oxidative phosphorylation and drive compensatory changes in the cell to use glycolysis to meet the cells' energy demands) were estimated using the XF Glycolytic Rate Assay Report Generator. Data are shown as mean±SD and analyzed with a t- test. ** p ≤ 0.01, **** p ≤ 0.0001 when compared with respective controls.

[0009] FIG. 6 shows a comparison of metabolic responses of cells of cervical cancer cell lines C-33A, Ca Ski, and SiHa treated with (+ or open circle) SHetA2 and without (- or closed circle) SHetA2. (A) The energetic map of the cervical cancer cells was assessed by XF Glycolytic ATP production rate assay report. (B) OCR / ECAR ratio was assessed, C-33A (lefthand bars), Ca Ski (center bars) and SiHa (righthand bars). (C) Mitochondrial / glycolytic  ATP ratios, C-33A (lefthand bars), Ca Ski (center bars) and SiHa (righthand bars) were calculated. **** p ≤ 0.0001 when compared with respective control.

[0010] FIG.7 shows that viability of cervical cancer cells decreases with increasing SHetA2 concentration. Representative dose-response curves and IC50values of (A) C-33A, (B) Ca Ski, and (C) SiHa cervical cancer cells. Cells were treated with SHetA2 (0-10 µM) for 72 hours in galactose vs. glucose media and assessed for metabolic viability using an MTT assay.

[0011] FIG.8 shows that inhibition of glycolysis by the glycolytic inhibitor 2-deoxyglucose (2-DG) increases sensitivity to SHetA2. Representative dose-response curves and IC50values of (A) C-33A, (B) Ca Ski, and (C) SiHa cervical cancer cells. Cells were treated with various doses of SHetA2 or a combination of SHetA2 and 0.25 mM of 2-DG for 72 hours.

[0012] FIG. 9 shows (A) isobolograms of SHetA2 and 2-DG combinations of Ca Ski cervical cancer cells treated with doses of SHetA2 and 2-DG. The graph shows the synergistic effect of SHetA2 and 2-DG in Caski cells. In (B) Ca Ski cervical cancer cells are treated with different concentrations of the SHetA2 alone or in combination with 2-DG then are analyzed using a 2-D ATP assay. In (C) Ca Ski cervical cancer cells are treated with different concentrations of the SHetA2 alone or in combination with 2-DG (0.5 mM and 1.0 mM) then are analyzed using a spheroid 3-D ATP assay.

[0013] FIG. 10 shows (A) isobolograms of SHetA2 and 2-DG combinations of SiHa cervical cancer cells treated with doses of SHetA2 and 2-DG. The graph shows the synergistic effect of SHetA2 and 2-DG in SiHa cells. In (B) SiHa cervical cancer cells are treated with different concentrations of the SHetA2 alone or in combination with 2-DG then are analyzed using a 2-D ATP assay. In (C) SiHa cervical cancer cells are treated with different concentrations of the SHetA2 alone or in combination with 2-DG (0.5 mM and 1.0 mM) then are analyzed using a spheroid 3-D ATP assay.

[0014] FIG. 11 shows in representative immunofluorescence images that SHetA2-induced DNA damage is not prevented by 2-DG and SHetA2 disrupts hsc70 / AIF complexes in Ca Ski cervical cancer cells. Cells were treated with either 10 µM SHetA2, or 2-DG or their combination (SHetA2+2-DG) for 24 hours and stained with MitoTracker™ Red CMXRos, γH2AX (green) and DAPI (blue) followed by confocal microscopy to check protein expression.

[0015] FIG. 12 shows in representative immunofluorescence images that SHetA2-induced DNA damage is not prevented by 2-DG and SHetA2 disrupts hsc70 / AIF complexes in SiHa cervical cancer cells. Cells were treated with either 10 µM SHetA2, or 2-DG or their combination (SHetA2+2-DG) for 24 hours and stained with MitoTracker™ Red CMXRos, γH2AX (green) and DAPI (blue) followed by confocal microscopy to check protein expression.

[0016] FIG.13 shows quantification of nuclear expression of γH2AX in the Ca Ski cells of FIG.11. Image J software was used to quantify.

[0017] FIG. 14 shows quantification of nuclear expression of γH2AX in the SiHa cells of FIG.12. Image J software was used to quantify.

[0018] FIG.15 shows that 2-DG enhances SHetA2 efficacy in SiHa xenograft model: Effect of SHetA2, 2-DG and combination treatment on average tumor volume (A), tumor weight (B) and body weight (C) in SiHa xenograft tumor bearing mice. * p ≤ 0.05, ** p ≤ 0.01 when compared with respective control.

[0019] FIG. 16 shows representative images of H&E staining on liver, kidney and spleen tissue, and -γH2AX IHC staining on tumor tissue collected from mice in different treatment group.

[0020] FIG. 17 shows quantification of γH2AX staining by image J software. * p ≤ 0.05, ** p ≤ 0.01 when compared with respective control.

[0021] The following abbreviations may be used herein: AIF: Apoptosis inducing factor, 6-AN: 6-aminonicotinamide, ANOVA: Analysis of Variance, ATP: Adenosine triphosphate, BNBZ: benitrobenrazide, 3-BP: 3-Bromopyruvate, BSA: Bovine serum albumin, BTF3: Basic transcription factor 3, CCRT: Concurrent Chemotherapy with Radiotherapy, CI: Combination index, DAPI: 4’,6-diamidino-2-phenylindole, DCA: Dichloroacetate, 2-DG: 2-deoxy-D-glucose, 2-DGP6: 2-deoxy-D-glucose-6-phosphate, DMEM: Dulbecco’s Modified Eagle Medium, DMSO: Dimethylsulfoxide, DRI: Dose-reduction index, ECAR: Extracellular acidification rate, ED: Effective dose, ED50: Effective dose in 50% of treated population, ED75: Effective dose in 75% of treated population, ED90: Effective dose in 90% of treated population, ED95: Effective dose in 95% of treated population, FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone, FDA: Food and Drug Administration, Flex-het: Flexible heteroarotinoid, FtsJ3: FtsJ RNA 2’-O-methyltransferase 3, GH: glycolytic inhibitor, GOG: Gynecologic Oncology Group,  GOT2: Glutamic-oxaloacetic transaminase 2, GP-2250: 1,4,5-oxathiazandioxide-4,4, G6PD: glucose-6-phosphate dehydrogenase, Grp78: glucose-regulated protein 78, Het: heteroarotinoid, Hsc70: heat shock cognate 70, HSP70: 70 kD heat shock protein, HGSOC: High-grade serous ovarian cancer, hFTSECs: Human fallopian tube Secretory epithelial cells, HK2: Hexokinase 2, HR-HPV: High-risk human papillomavirus, IACUC: Institutional Animal Care and Use Committee, IC: Inhibitory concentration, IC50: Half maximal inhibitory concentration, LND: Lonidamine, M: Molar, MET: Metformin, mg: Milligram, mM: Millimolar, MOTD: (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol, M-PER: Mammalian Protein Extraction Reagent, MTT: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, OCR: Oxygen consumption rate, OD: Optical density, OS: Overall Survival, OT: oxythiamine, OUHSC: University of Oklahoma Health Sciences Center, OxPhos: Oxidative phosphorylation; PER: Proton efflux rate, PFK15: 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), 3PO: (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one), PUF60: Poly (U) binding splicing factor 60, RAID: Rapid Access to Intervention Development, RAPID: Rapid Access to Preventive Intervention Development, ROS: Reactive oxygen species, SD: Standard deviation, SEM: Standard deviation of the mean, SHetA2: (4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl]amino]methane- thione, TCGA: The Cancer Genome Atlas, TMA: Tumor Microarray, TP53: Tumor protein 53 gene, VARS1: valyl-tRNA synthetase 1, and wt / wt: Weight / weight. DETAILED DESCRIPTION

[0022] The present disclosure is directed to combination treatments for improved anti- cancer therapies. The drug combinations include at least one heteroarotinoid and at least one  glycolytic inhibitor. In at least certain non-limiting embodiments, the drugs act synergistically against cancer viability.

[0023] Before further detailed description of various embodiments of the compositions and methods of use thereof of the present disclosure, it is to be understood that the present disclosure is not limited in application to the details of methods and compositions as set forth in the following description. The description provided herein is intended for purposes of illustration only and is not intended to be construed in a limiting sense. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that various embodiments of the present disclosure may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. It is intended that all alternatives, substitutions, modifications and equivalents apparent to those having ordinary skill in the art are included within the scope of the present disclosure as defined herein. Thus, the examples described below, which include particular embodiments, will serve to illustrate the practice of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as of the principles and conceptual aspects of the inventive concepts. Thus, while the compositions and methods of the present disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the inventive concepts disclosed herein.

[0024] Each patent, published patent application, and non-patent publication referenced in any portion of this application, including but not limited to U.S. Patent No. 11,534,397; U.S. Ser. No. 18 / 145,334, filed December 22, 2022; U.S. Ser. No.18 / 145,334, filed December 22, 2022; U.S. Ser. No. 17 / 276,032, filed March 12, 2021; International Application No. PCT / US2019 / 050775, filed Sept.12, 2019; U.S. Provisional Application Ser. No.63 / 578,180  filed August 23, 2023, and U.S. Provisional Application Serial No.62 / 730,345, filed Sept.12, 2018, is expressly incorporated herein by reference in its entirety to the same extent as if the individual patent, or published patent application, or non-patent publication was specifically and individually indicated to be incorporated by reference.

[0025] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those having ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0026] As utilized in accordance with the methods and compositions of the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0027] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or when the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, or any integer inclusive therein. The term “at least one” may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0028] As used herein, all numerical values or ranges include fractions of the values and integers within such ranges and fractions of the integers within such ranges unless the context clearly indicates otherwise. Thus, to illustrate, reference to a numerical range, such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so forth. Reference to a series of ranges includes ranges which combine the values of the boundaries of different ranges within the series. Thus, to illustrate reference to a series of ranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-  200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, includes ranges of 1-20, 10- 50, 50-100, 100-500, and 500-1,000, for example.

[0029] As noted above, any numerical range listed or described herein is intended to include, implicitly or explicitly, any number or sub-range within the range, particularly all integers, including the end points, and is to be considered as having been so stated. For example, "a range from 1.0 to 10.0" is to be read as indicating each possible number, including integers and fractions, along the continuum between and including 1.0 and 10.0, that is, having a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 10.0, such as, for example, 3.25 to 8.65. Any maximum numerical limitation recited herein is intended to include all lower numerical limitations subsumed therein, and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range subsumed within the ranges expressly recited herein. Thus, even if a particular data point within the range is not explicitly identified or specifically referred to, it is to be understood that any data points within the range are to be considered to have been specified, and that the inventor(s) possessed knowledge of the entire range and the points within the range.

[0030] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0031] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0032] Throughout this application, the terms “about” or “approximately” are used to indicate that a value includes the inherent variation of error for the composition, the method used to administer the composition, or the variation that exists among the study subjects. As used herein the qualifiers “about” or “approximately” are intended to include not only the exact  value, amount, degree, orientation, or other qualified characteristic or value, but are intended to include some slight variations due to measuring error, manufacturing tolerances, observer error, and combinations thereof, for example. The term “about” or “approximately”, where used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass, for example, variations of ± 20%, or ± 10%, or ± 5%, or ± 1%, or ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. As used herein, the term “substantially” means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, the term “substantially” means that the subsequently described event or circumstance occurs at least 80% of the time, at least 90% of the time, at least 91% of the time, at least 92% of the time, at least 93% of the time, at least 94% of the time, at least 95% of the time, at least 96% of the time, at least 97% of the time, at least 98% of the time, or at least 99% of the time.

[0033] As used herein any reference to “one embodiment” or “an embodiment” means that a particular element, feature, composition, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0034] Where used herein, the pronoun “we” is intended to refer to all persons involved in a particular aspect of the investigation disclosed herein and as such may include non-inventor laboratory assistants and collaborators working under the supervision of the inventor(s).

[0035] The term “mutant” or “variant” is intended to refer to a protein, peptide, nucleic acid or organism which has at least one amino acid or nucleotide which is different from the wild type version of the protein, peptide, nucleic acid, or organism and includes, but is not limited to, point substitutions, multiple contiguous or non-contiguous substitutions, chimeras, or fusion proteins, and the nucleic acids which encode them. Examples of conservative amino acid substitutions include, but are not limited to, substitutions made within the same group such as within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as  glycine, alanine, serine, threonine, methionine). Other examples of possible substitutions are described below.

[0036] The term “pharmaceutically acceptable” refers to compounds and compositions which are suitable for administration to humans and / or animals without undue adverse side effects such as toxicity, irritation and / or allergic response commensurate with a reasonable benefit / risk ratio.

[0037] As used herein, “pure” or “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other object species in the composition thereof), and particularly a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more particularly more than about 85%, more than about 90%, more than about 95%, or more than about 99%. The term “pure” or “substantially pure” also refers to preparations where the object species (e.g., the active agent) is at least 60% (w / w) pure, or at least 70% (w / w) pure, or at least 75% (w / w) pure, or at least 80% (w / w) pure, or at least 85% (w / w) pure, or at least 90% (w / w) pure, or at least 92% (w / w) pure, or at least 95% (w / w) pure, or at least 96% (w / w) pure, or at least 97% (w / w) pure, or at least 98% (w / w) pure, or at least 99% (w / w) pure, or 100% (w / w) pure.

[0038] The terms “subject” and “patient” are used interchangeably herein and will be understood to refer to a warm-blooded animal, particularly a mammal. Non-limiting examples of animals within the scope and meaning of this term include dogs, cats, rabbits, rats, mice, guinea pigs, chinchillas, hamsters, ferrets, horses, pigs, goats, cattle, sheep, zoo animals, camels, llamas, non-human primates, including Old and New World monkeys and non-human primates (e.g., cynomolgus macaques, chimpanzees, rhesus monkeys, orangutans, and baboons), and humans.

[0039] The term “active agent” where used herein is intended to refer to a substance which possesses a biological activity relevant to the present disclosure, and particularly refers to therapeutic and diagnostic substances which may be used in methods described in the present disclosure. By “biologically active” is meant the ability to modify the physiological system of a cell, tissue, or organism without reference to how the active agent has its physiological effects. Where used herein, unless otherwise noted, the term “active agent” includes pharmaceutically-acceptable salts, hydrates, solvates, and amorphous solids thereof. Particular  examples of active agents of the present disclosure include heteroarotinoids and glycolytic inhibitors as defined elsewhere herein.

[0040] The term “heteroarotinoid” as used herein refers to a retinoid compound which has a heteroatom substitution (e.g., S, N, O) on the ring structure. The term flexible heteroarotinoid, or “Flex-Het,” refers to a heteroarotinoid (e.g., SHetA2) having a flexible linker between the heteroatom ring and the aryl ring, such as a urea or thiourea linker.

[0041] The term "glycolysis" as used herein refers to the enzymatic oxidation of glucose by a cell. During glycolysis, glucose is broken down into either lactate or pyruvate. Under aerobic conditions, the main product is pyruvate. When oxygen is depleted, the main glycolytic product is lactate. Examples of enzymes that are involved in glycolysis include, for example, hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, phosphopyruvate hydratase, and pyruvate kinase. Another “glycolytic” pathway is the Pentose Phosphate Pathway (PPP) in which glucose-6-phosphate produced from glucose using hexokinase is acted on by glucose-6-phosphate dehydrogenase (G6PD) to ultimately produce ribose-5-phosphate for nucleotide formation.

[0042] The term "glycolytic inhibitor” as used herein refers to a substance that inhibits or negatively alters the enzymatic glycolysis of glucose, wherein the inhibitor directly acts on an enzyme (e.g., see above) or coenzyme or cofactor that is involved in glycolysis or in the PPP, or acts on end products of glycolysis or the PPP. Examples of such glycolytic inhibitors may include but are not limited to 2-DG, acetylated or halogenated forms of 2- DG, 3-BP, PFK15, 3PO, DCA, LND, 6-AN, BNBZ, GP-2250, MOTD, and Metformin, as further described elsewhere herein.

[0043] “Treatment” refers to therapeutic treatments. “Prevention” refers to prophylactic or preventative treatment measures or reducing the onset of a condition or disease. The term “treating” refers to administering the composition to a subject for therapeutic purposes and / or for prevention. Non-limiting examples of modes of administration include inhalation, oral, topical, retrobulbar, subconjunctival, transdermal, parenteral, subcutaneous, intranasal, intramuscular, intraperitoneal, intravitreal, and intravenous routes, including both local and systemic applications. In addition, the compositions of the present disclosure may be designed to provide delayed, controlled, extended, and / or sustained release using formulation techniques which are well known in the art.

[0044] The terms “therapeutic composition” and “pharmaceutical composition” refer to an active agent-containing composition that may be administered to a subject by any method  known in the art or otherwise contemplated herein, wherein administration of the composition brings about a therapeutic effect as described elsewhere herein. In addition, the compositions of the present disclosure may be designed to provide delayed, controlled, extended, and / or sustained release using formulation techniques which are well known in the art.

[0045] Where used herein the term “drug combination” or “combo” refers to a combination of drugs (compounds) which can be conjointly administered, such as a heteroarotinoid and a glycolytic inhibitor.

[0046] As used herein the terms “conjoint,” “conjointly,” “conjointly administered,” or “conjoint administration” refers to any form of administration of a combination of two or more different therapeutic compounds (also referred to herein as drugs or active agents) such that the second compound is administered while the previously administered therapeutic compound is still effective in the body, whereby the two or more compounds are simultaneously active in the patient, enabling a synergistic interaction of the compounds. For example, the different therapeutic compounds can be administered either together in the same formulation (i.e., as a physical mixture), or in separate formulations, either concomitantly (at the same time) or sequentially. When administered sequentially the different compounds may be administered immediately in succession, or separated by a suitable duration of time, as long as the active agents function together in a synergistic manner.

[0047] The term “effective amount” refers to an amount of an active agent which is sufficient to exhibit a detectable therapeutic effect without excessive adverse side effects (such as toxicity, irritation and allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner disclosed herein. The effective amount for a patient will depend upon the type of patient, the patient’s size and health, the nature and severity of the condition to be treated, the method of administration, the duration of treatment, the nature of concurrent therapy (if any), the specific formulations employed, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of ordinary skill in the art using routine experimentation based on the information provided herein.

[0048] The term "ameliorate" means a detectable or measurable improvement in a subject's condition, disease or symptom thereof. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the condition or disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease. A successful treatment outcome can lead to a "therapeutic effect," or  "benefit" of ameliorating, decreasing, reducing, inhibiting, suppressing, limiting, controlling or preventing the occurrence, frequency, severity, progression, or duration of a disease or condition, or consequences of the disease or condition in a subject.

[0049] A decrease or reduction in worsening, such as stabilizing the condition or disease, is also a successful treatment outcome. A therapeutic benefit therefore need not be complete ablation or reversal of the disease or condition, or any one, most or all adverse symptoms, complications, consequences or underlying causes associated with the disease or condition. Thus, a satisfactory endpoint may be achieved when there is an incremental improvement such as a partial decrease, reduction, inhibition, suppression, limit, control, or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal of the condition or disease (e.g., stabilizing), over a short or long duration of time (hours, days, weeks, months, etc.). Effectiveness of a method or use, such as a treatment that provides a potential therapeutic benefit or improvement of a condition or disease, can be ascertained by various methods and testing assays.

[0050] Turning now to the inventive concepts, cervical cancer is the second most common cancer among women and fourth in terms of mortality across genders. For locally advanced cervical cancer, the optimal therapy is CCRT. These modalities are highly toxic and cause significant morbidity. Moreover, patients with recurrent and metastatic cervical cancer are very resistant to chemotherapy and can hardly be cured, with a 1-year survival rate of less than 20%. Although recent addition of FDA-approved immune-based therapy has shown improved survival, cervical cancer lags behind most other cancers in having molecular-based drugs available for therapeutic use

[0051] We developed a non-toxic investigational new agent, SHetA2, currently in Phase 1 trial for treatment of advanced and recurrent gynecologic cancers (clinicaltrials.gov: NCT04928508). SHetA2 kills cancer cells without harming healthy cells by binding to HSP70 proteins (mortalin, Grp78 and hsc70). Elevated HSP70s in cancer cells represent cancer- selective SHetA2 targets that are only present at low levels in healthy cells, which explains the lack of toxicity noted for SHetA2 in extensive preclinical studies. HSP70s become elevated during carcinogenesis to protect the developing cancer cells from the deleterious effects of elevated oncoproteins. The elevated HSP70s bind to elevated oncogenic client proteins to control their stability and cellular localization in a way that allows the carcinogenic oncoprotein activities, such as elevated cell cycle progression, while repressing the cell-death inducing oncoprotein activities. SHetA2 binding to mortalin, hsc70 and Grp78 causes release of their  client proteins, which then become susceptible to protein degradation and intracellular relocation.

[0052] We established the potent anti-cancerous activity of our new investigational drug, SHetA2 against cervical cancer. Further mechanistic studies demonstrated that SHetA2 treatment leads to metabolic reprogramming by inhibition of oxidative phosphorylation (OxPhos) and upregulation of glycolysis. Inhibition of glycolysis by (for example but not by way of limitation) a hexokinase inhibitor such as 2-DG increased SHetA2 sensitivity in vitro and in vivo in a cervical cancer cell xenograft model.

[0053] 2-DG is one non-limiting example of a glycolytic inhibitor that may utilized in accordance with the present disclosure. 2-DG is a hexokinase inhibitor and has multiple attributes as a candidate therapeutic agent. There is substantial clinical information about 2- DG and its 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) analog as they have been extensively utilized to enhance diagnostic imaging. Glucose, 2-DG, and FDG have increased uptake in cancer tissues due to the elevation of glucose transporters in cancer cells, which allows for the imaging of malignancies and metabolically active tissues in contrast to nonmalignant tissues. Also, 2-DG is reasonably well-tolerated in cancer patients. In a phase I study of oral 2-DG in patients with solid tumors, 45 mg / kg 2-DG given on a daily schedule on the first two weeks of a three-week cycle was defined as the recommended dose based on cardiac QTc prolongation occurring in two of 4 patients at the 60 mg / kg dose level, and none of the 5 patients treated at the 45 mg / kg dose level.2-DG also has considerable potential for use in combination therapies. In preclinical studies, 2-DG sensitized cancer cells to multiple types of chemotherapeutic agents, and paclitaxel enhanced the uptake of 2-DG. 2-DG sensitized pancreatic cancer cells and tumors to inhibition of MEK, the kinase immediately upstream of MAPK3 in the RAS- induced kinase activation cascade. Thus, in a non-limiting embodiment, the present disclosure is directed to the use of 2-DG with heteroarotinoids that induce glycolysis for improving the cancer therapeutic efficacies without significantly increasing toxicity.

[0054] Therefore, certain non-limiting embodiments of the present disclosure are directed to therapeutic drug combinations that include at least a first active agent selected from at least one heteroarotinoid, particularly a flexible heteroarotinoid (such as are further described  below); and at least a second active agent comprising at least one glycolytic inhibitor as defined elsewhere herein.

[0055] Where used herein the terms “heteroarotinoid,” and specific examples thereof, and “glycolytic inhibitor,” and specific examples thereof, are intended to include pharmaceutically- acceptable salts, hydrates, solvates, and amorphous solids thereof.

[0056] Particular examples of heteroarotinoids include flexible heteroarotinoids (i.e., hets having a urea or thiourea linker (also referred to herein as “Flex-Hets”)), such as, but not limited to, any heteroarotinoid disclosed in U.S. Patent Nos.6,586,460 (see, for example, Columns 2- 5 thereof) and 7,612,107 (see, for example, Columns 7-9 thereof). Non-limiting examples of heteroarotinoids that may be used herein include SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, NHet90, and other heteroarotinoid compounds shown in Table 1, compounds 1-3 shown in Table 2, heteroarotinoid compounds shown in Table 3, heteroarotinoid compounds shown in Table 4, heteroarotinoid compounds shown in Table 5, heteroarotinoid compounds shown in Table 6, heteroarotinoid compounds shown in Table 7, and heteroarotinoid compounds shown in Table 8.  Table 1. Heteroarotinoids (Benbrook et al., 1997*) *D.Activity and Decreased Toxicity,” J. Med. Chem., 1977, 40 (3567-3583).  Table 2. Nitrogen Heteroarotinoids (Dhar et al., 1999*) *A. DharInteractions of Nitrogen Heteroarotinoids,” J. Med. Chem., 1999, 42 (3602-3614).  Table 3. Heteroarotinoids (Zacheis et al., 1997*) *D.Vitro and in Vivo Through Both RAR and RXR Retinoic Acid Receptors,” J. Med. Chem., 1999, 42 (4434- 4445).  Table 4. Heteroarotinoids (Brown et al., 2004*) *C. W.bovis BCG,” J. Med. Chem., 2004, 47 (1008-1017).  Table 5. Heteroarotinoids (Le et al., 2007*)Table 6. Heteroarotinoids (Liu et al., 2004*)*S. Liu et al., “Synthesis of Flexible Sulfur-Containing Heteroarotinoids That Induce Apoptosis and Reactive Oxygen Species with Discrimination between Malignant and Benign Cells,” J. Med. Chem., 2004, 47 (999-1007).  Table 7. Heteroarotinoids (Gnanasekaran et al., 2015*)*K. K.Flex-Het scaffolds against the human ovarian A2780 cancer cell line,” European J. Med. Chem., 2015, 96 (209- 217).  Table 8. Heteroarotinoids (Liu et al., 2009*) *T. Liu et al.,kidney cancer,” Mol. Cancer Ther., 2009, 8(5), (1227- .

[0057] Particular examples of glycolytic inhibitors that can be utilized in accordance with the present disclosure include 2-deoxy-D-glucose (2-DG), 2-fluoro-2-deoxy-D-glucose (2- FG), 2-chloro-2-deoxy-D-glucose (2-CG), 2-bromo-2-deoxy-D-glucose (2-BG), acetylated derivatives of 2-DG, (e.g., WP1208, WP1124, WP1125, WP1209, WP1210, WP1122, and WP1211, see Table 9); 3-bromopyruvate (3-BP); 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1- one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6-aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5- oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-6β-methoxyergosta-7,9(11),22- triene-3β,5α-diol (MOTD); and metformin (MET), and combinations of the above.

[0058] Table 9 shows several non-limiting examples of acetylated derivatives of 2-DG that may be used in combination with heteroarotinoids in accordance with the inventive concepts of the present disclosure.  Table 9. Acetylated and halogenated derivatives of 2-DG*based antimetabolites. Part 4: Facile synthesis of mono- and di-acetates of 2-deoxy-D-glucose prodrugs as potent useful antimetabolites. Carbohydr. Res.2023, 531, 108861. Pajak, Siwiak, Soltyka, A. Priebe, Zielinski, Fokt, Ziemniak, Jaskiewicz, Borowski, Domoradzki, W. Priebe. Int. J. Mol. Sci., 2020 Jan; 21(1): 234.

[0059] Particular therapeutic drug combinations of the present disclosure include, but are not limited to, at least one first active agent selected from at least one heteroarotinoid, particularly (but not limited to) a flex-het, and at least one glycolytic inhibitor such as described herein. Non-limiting examples of such drug combinations are shown in Tables 10-12. Table 10. Non-Limiting Heteroarotinoid and Glycolytic Inhibitor Drug Combinations Het+2-DG Het+2-DG Het+2-DG Het+2-DG Het+LND Het+3-BP Het+DCA Combo Combo Combo Combo Combo Combo Combo + + + + +  SHetD4+ SHetD4+ SHetD4+ SHetD4+ SHetD4+ SHetD4+ SHetD4+ 2-DG 2-DG 2-DG 2-DG LND 3-BP DCA SHetD5+ SHetD5+ SHetD5+ SHetD5+ SHetD5+ SHetD5+ SHetD5+ + + + + + +Table 11. Non-Limiting Heteroarotinoid and Glycolytic Inhibitor Drug Combinations Het+6AN Het+BNBZ Het+GP-2250 Het+MOTD Het+MET Het+3PO Het+PFK15 Combo Combo Combo Combo Combo Combo Combo  Table 12. Non-Limiting Heteroarotinoid and Glycolytic Inhibitor Drug Combinations Het+WP1208 Het+WP1124 Het+WP1125 Het+WP1209 Het+WP1210 Het+WP1211 Het+WP1122 Combo Combo Combo Combo Combo Combo Combo SHetA2+ SHetA2+ SHetA2+ SHetA2+ SHetA2+ SHetA2+ SHetA2+

[0060] LND is a derivative of indazole-3-carboxylic acid and is known to inhibit aerobic glycolysis and energy metabolism selectively in tumor cells. LND was previously evaluated in Phase II and Phase III trials targeting lung cancer and was shown to be safe, but to have limited efficacy. LND has undergone clinical trials in combination with standard-of-care chemotherapeutics for multiple cancers (including breast cancer, glioblastoma multiforme, ovarian cancer, and lung cancer, benign prostatic hyperplasia).

[0061] 3-BP is an inhibitor of hexokinase shown to abolish ATP production and cause severe depletion of cellular ATP. Like 2-DG, 3-BP also exhibits potent cytotoxic activity against cancer cells with mitochondrial respiratory defects and cells in a hypoxic environment. 3-BP is effective at the concentration of 100 μM, which is more potent than 2-DG (effective in mM range). Combinations of 3-BP with an mTOR inhibitor seems to have synergistic effects on leukemia and lymphoma cells. Animal studies showed that 3-BP has significant in vivo therapeutic activity against liver cancer when the compound was given by local infusion and seems to inhibit metastasis when given intravenously.

[0062] 1,4,5-oxathiazandioxide-4,4 (GP-2250) is an oxathiazinine derivative that reduces ATP production in cells by inhibiting hexokinase 2 and glyceraldehyde-3-phosphate leading to inhibition of cancer cell culture and tumor growth.

[0063] 6-aminonicotinamide (6AN) inhibits the pentose phosphate pathway by inhibiting G6PD.6AN exhibits anticancer activity in vitro, causes oxidative stress, and sensitizes cells to anticancer agents and radiation; however, it also causes neurotoxicity and other toxic side effects.

[0064] Benitrobenrazide (BNBZ) binds to HK2 and inhibits its enzymatic activity, reduces glycolysis and growth in cancer cells and reduces tumor growth.

[0065] (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD) from Ganoderma sinense binds to and inhibits the activity of HK2 and is cytotoxic to cancer cells.

[0066] Metformin (MET) inhibits hexokinase and reduces glucose metabolism in cancer cells and growth of tumors.

[0067] Imatinib (GLEEVEC; IMA) and Oxythiamine (OT) are non-specific inhibitors of glycolysis that may be used with the Hets of the present disclosure. Imatinib is a tyrosine kinase inhibitor designed to specifically target BCR-ABL protein, which is responsible for the development of chronic myeloid leukemia (CML). The Bcr-Abl oncogene is a fusion DNA sequence created by chromosome translocation and codes for a constitutively active tyrosine kinase fusion protein. The BCR-ABL-positive cells express the high-affinity glucose transporter (GLUT-1) and exhibit increased glucose uptake. Imatinib treatment decreases the activity of both hexokinase and G6PD in leukemia cells, leading to suppression of aerobic glycolysis. A decrease in G6PD activity would lead to lower glucose flow into the pentose phosphate pathway, and thus deprives transformed cells of metabolic intermediates for ATP generation and substrates for macromolecule synthesis. Although imatinib is an antileukemia drug, its ability to suppress aerobic glycolysis may make it useful for the treatment of certain solid tumors. OT is a thiamine antagonist and inhibits transketolase and pyruvate dehydrogenase.

[0068] Where used herein the term cancer, cancer cell, or tumor, can be used in reference to various cancers including, but not limited to cervical squamous cell carcinoma, endocervical adenocarcinoma, ovarian serous cystadenocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, lung squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, esophageal carcinoma, stomach adenocarcinoma, uterine carcinosarcoma, uterine corpus endometrial carcinoma, liver hepatocellular carcinoma,  kidney renal papillary cell carcinoma cholangiocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, mesothelioma, ovarian cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, and any other cancer which comprises one or more mutations in TP53 gene or which is driven by cyclin D1 / cdk4 / 6 complex.

[0069] In other embodiments, the term cancer, cancer cell, or tumor, can be used in reference to various cancers including, but not limited to oral cancer, pharyngeal cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer, peritoneal cancer, uterine cancer, gastrointestinal cancer, central or peripheral nervous system tissue cancer, an endocrine cancer, neuroendocrine cancer, glioma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, renal cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumors, thyroid cancer, parathyroid cancer, pituitary tumors, adrenal gland tumors, osteogenic sarcoma tumors, multiple neuroendocrine type I and type II tumors, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer.

[0070] In other embodiments, the term cancer, cancer cell, or tumor, can be used in reference to various cancers including, but not limited to breast cancer (e.g., estrogen receptor positive or negative, progesterone receptor positive or negative, HER-2 positive or negative, triple-negative breast cancer, or BRCAl and / or BRCA2 positive or negative), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), endometrial cancer, glioblastoma, B-cell malignancies, biliary tract cancer, bone cancer, choriocarcinoma, connective tissue cancer, cancers of the digestive system, gallbladder cancer, hepatocellular carcinoma, intra-epithelial neoplasm, kidney cancer, liver cancer, lymphoma, skin cancer (e.g., melanoma and basal cell carcinoma), neuroblastoma, mesothelioma, neuroglioma, oral cavity cancer, pediatric cancer, pancreatic endocrine tumors, pituitary adenoma, thymoma, renal cell carcinoma, salivary gland cancer, sarcoma (e.g., Ewing's sarcoma, fibrosarcoma, and rhabdomyosarcoma), small bowel cancer, ureteral cancer, cancers of the urinary system, and hematological cancers (e.g., acute myeloid leukemia and multiple myeloma).

[0071] Exemplary solid cancerous tumors that can be treated with the active agents of the present disclosure include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, liver, gallbladder, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, uterus, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias,  lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, thyroid cancer, and melanoma.

[0072] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor;  nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; B-cell lymphoma; low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0073] The active agents of the present disclosure may be combined with one or more pharmaceutically-acceptable excipients, including carriers, vehicles, diluents, and adjuvants which may improve solubility, deliverability, dispersion, stability, and / or conformational integrity of the compounds or conjugates thereof.

[0074] “Pharmaceutically acceptable salts” means salts of active agent compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include (but are not limited to) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,  nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′- methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1- carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p- toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include (but are not limited to) base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include (but are not limited to) sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include (but are not limited to) ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of the present disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional non-limiting examples of pharmaceutically acceptable salts and their methods of preparation and use are shown in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0075] The term “conjoint” or "coadministration" refers to administration of two or more active agents, e.g., a cardiac-targeted composition as described herein and another active agent. The timing of coadministration depends in part on the combination and compositions administered and can include administration at the same time, just prior to, or just after the administration of one or more additional therapies. “Coadministration” is meant to include simultaneous or sequential administration of the compound and / or composition individually or in combination. Thus, the preparations can also be combined, when desired, with other active substances (e.g., to reduce metabolic degradation). For example, the compositions described  herein can be used in combination with one another, or with other active agents known to be useful in treating MI, and co-occurring conditions thereof.

[0076] The active agents of the present disclosure may be present in the pharmaceutical compositions (alone or in combination) at any concentration that allows the pharmaceutical composition to function in accordance with the present disclosure; for example, but not by way of limitation, the compound(s) may be present in a carrier, diluent, or buffer solution in a wt / wt or vol / vol range having a lower level selected from 0.00001%, 0.0001%, 0.005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% and 2.0%; and an upper level selected from 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Non-limiting examples of particular wt / wt or vol / vol ranges include a range of from about 0.0001% to about 95%, a range of from about 0.001% to about 75%; a range of from about 0.005% to about 50%; a range of from about 0.01% to about 40%; a range of from about 0.05% to about 35%; a range of from about 0.1% to about 30%; a range of from about 0.1% to about 25%; a range of from about 0.1% to about 20%; a range of from about 1% to about 15%; a range of from about 2% to about 12%; a range of from about 5% to about 10%; and the like. Any other range that includes a lower level selected from the above-listed lower-level concentrations and an upper level selected from the above-listed upper-level concentrations also falls within the scope of the present disclosure. Percentages used herein may be weight percentages (wt%) or volume percentages (vol%).

[0077] In certain non-limiting embodiments, an effective amount or therapeutic dosage of a pharmaceutical composition of the present disclosure contains, sufficient active agent to deliver from about 0.001 μg / kg to about 100 mg / kg (weight of active agent / body weight of the subject). For example, the composition will deliver about 0.01 μg / kg to about 50 mg / kg, and more particularly about 0.1 μg / kg to about 10 mg / kg, and more particularly about 1 μg / kg to about 1 mg / kg. Practice of a method of the present disclosure may comprise administering to a subject an effective amount of the active agent in any suitable systemic and / or local formulation, in an amount effective to deliver the therapeutic dosage of the active agent. In certain embodiments, an effective dosage may be, in a range of about 1 μg / kg to about 1 mg / kg of the active agent.

[0078] In certain non-limiting embodiments, an effective amount or therapeutic dosage of a pharmaceutical composition of the present disclosure contains at least one heteroarotinoid (e.g., SHetA2) in a dose range that will result in 0.01 micromolar to 100 millimolar blood or  tissue levels, and at least one GH (e.g., 2-DG) in a concentration in a dose range that will result in 0.01 micromolar to 100 millimolar blood or tissue levels. In certain non-limiting embodiments, an effective amount or therapeutic dosage of a pharmaceutical composition of the present disclosure contains the Het in a concentration in a dose range that will result in 0.01 micromolar to 100 millimolar, and the GH in a dose range that will result in 0.01 micromolar to 100 millimolar blood or tissue levels.

[0079] In non-limiting embodiments, the synergistic ratio of the compositions of the first active agent (a Het) and the second active agent (a GH) may be in a range from 50,000:1 wt / wt to 1:50,000 wt / wt, 40,000:1 wt / wt to 1:40,000 wt / wt, 30,000:1 wt / wt to 1:30,000, 20,000:1 wt / wt to 1:20,000 wt / wt, 10,000:1 wt / wt to 1:10,000 wt / wt, 5,000:1 wt / wt to 1:5,000 wt / wt, 1,000:1 wt / wt to 1:1,000 wt / wt, 500:1 wt / wt to 1:500 wt / wt, 100:1 wt / wt to 1:100 wt / wt, 50:1 wt / wt to 1:50 wt / wt, 45:1 wt / wt to 1:45 wt / wt, 40:1 wt / wt to 1:40 wt / wt, 35:1 wt / wt to 1:35 wt / wt, 30:1 wt / wt to 1:30 wt / wt, 25:1wt / wt to 1:25 wt / wt, e.g., 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25 or may be in any range bounded by the ratios listed above.

[0080] In a particular (but non-limiting) embodiment, a ratio of 0.1 µM Het (e.g., SHetA2) to 4 mM GH (e.g., 2-DG) provides a 1:40,000 wt / wt ratio of heteroarotinoid:glycolytic inhibitor.

[0081] The term "synergistic" or “synergistic effect” or “synergistic interaction” as used herein refers to a therapeutic combination which is more effective than the additive effects of the two or more single active agents. A “synergistic ratio” is a ratio of two compounds which results in a synergistic effect. A determination of a synergistic interaction between the active agents described herein may be based on the results obtained from the assays described herein. The results of these assays can be analyzed using the Chou and Talalay combination method (Chou TC, Talalay P. “Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.” Adv Enzyme Regul.1984;22: 27-55) and Dose-Effect Analysis with CalcuSyn software in order to obtain a Combination Index. The combinations provided herein have been evaluated in several assay systems, and the data can be analyzed utilizing a standard program for quantifying synergism, additivism, and antagonism among anticancer agents. An example program is that described by Chou and Talalay, in "New Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter 2. Combination  Index values less than 0.9 indicate synergy, values greater than 1.2 indicate antagonism and values between 0.9 to 1.1 indicate additive effects (e.g., see Table 4 below). The combination therapy may provide "synergy" and prove "synergistic", i.e., the effect achieved when the active agents used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active agents are: (1) co- formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered in succession (“alternation therapy”) or in parallel as separate formulations; or (3) by some other effective regimen. When delivered in successive administrations, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[0082] Certain non-limiting embodiments of the present disclosure are directed to a method that comprises administering to a subject in need thereof any of the pharmaceutical compositions disclosed or otherwise contemplated herein.

[0083] The pharmaceutical compositions may be administered via any mechanisms disclosed herein or otherwise contemplatable by a person having ordinary skill in the art. In one non-limiting embodiment, the administration occurs via an inhaler, which aerosolizes the active agents.

[0084] The pharmaceutical compositions of the present disclosure may be administered for any purpose disclosed or otherwise contemplated herein, as well as for any purpose within the purview of a person having ordinary skill in the art. In one non-limiting embodiment, the pharmaceutical compositions are administered in a method of treating or reducing the occurrence of cancer. However, this treatment method is not to be construed as limiting of the present disclosure, and any diseases, disorders, or conditions disclosed herein or otherwise contemplatable by a person having ordinary skill in the art (given the subject application) which may derive a therapeutic effect by treatment with the compositions disclosed herein also fall within the scope of the methods of the present disclosure.

[0085] Practice of the methods of the present disclosure may comprise administering to a subject therapeutically effective amounts of each active agent in any suitable systemic and / or local formulation, in an amount effective to deliver the dosages listed herein. The dosage can be administered, for example but not by way of limitation, on a one-time basis, or administered at multiple times (for example but not by way of limitation, from one to five times per day, or  once or twice per week), or continuously via a venous drip, depending on the desired therapeutic effect. In one non-limiting example of a therapeutic method of the present disclosure, each active agent is provided in an IV infusion in the range of from about 0.01 mg / kg to about 10 mg / kg of body weight once a day.

[0086] The compositions and dosage forms of the present disclosure can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight, and condition of the subject, the particular composition used, and the route of administration. In one non-limiting embodiment, a single dose of the composition according to the disclosure is administered. In other non-limiting embodiments, multiple doses are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, or whether the composition is used for prophylactic or curative purposes. For example, in certain non-limiting embodiments, the composition is administered once per month, twice per month, three times per month, every other week, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily, twice a day, or three times a day. The duration of treatment (i.e., the period of time over which the composition is administered) can vary, depending on any of a variety of factors, e.g., subject response. For example, the composition can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more. Where used herein, unless otherwise indicated, the dosage amount refers to the amount of each active agent that is administered to the subject.

[0087] The dosage of an administered active agent for humans will vary depending upon factors such as (but not limited to) the patient's age, weight, height, sex, general medical condition, and previous medical history. In certain non-limiting embodiments, where each active agent is administered by injection or infusion, the recipient may be provided with a dosage of each active agent that is in the range of from about 1 mg to about 1000 mg, and it may be administered as a single infusion or multiple injections, although a lower or higher dosage also may be administered. In certain non-limiting embodiments, the dosage may be in the range of from about 25 mg to about 100 mg of the active agent per square meter (m2) of body surface area for a typical adult, although a lower or higher dosage also may be administered. Non-limiting examples of dosages of each active agent that may be administered to a human subject include, but are not  limited to, those in ranges of 1 to 1000 mg, 1 to 600 mg, 1 to 500 mg, 1 to 400 mg, 1 to 300 mg, 1 to 200 mg, 100 to 600 mg, 100 to 500 mg, 100 to 400 mg, 100 to 300 mg, 100 to 200 mg, 150 to 600 mg, 150 to 500 mg, 150 to 400 mg, 150 to 300 mg, 150 to 250 mg, 150 to 200 mg, 200 to 7500 mg, 200 to 600 mg, 200 to 500 mg, 200 to 400 mg, 200 to 300 mg, and 200 to 250 mg, or any subrange within any of the aforementioned ranges. Dosages may be repeated as needed, for example (but not by way of limitation), once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as (but not limited to) every other week for several months, or more frequently, such as twice weekly or by continuous infusion.

[0088] In certain non-limiting embodiments, the present disclosure is directed to a dosing regimen of each active agent comprising multiple dosing cycles (e.g., wherein the first dosing cycle is a step-up, fractionated dosing cycle). In some non-limiting embodiments, the dose of each active agent may range from 50 mg to 200 mg (e.g., from 50 mg to 175 mg, from 50 mg to 150 mg, from 50 mg to 125 mg, from 50 mg to 100 mg, from 50 mg to 75 mg, from 50 mg to 70 mg, from 52 mg to 100 mg, from 52 mg to 75 mg, from 50 mg to 180 mg, from 55 mg to 150 mg, from 55 mg to 100 mg, from 55 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; e.g., about 60 mg, ,or any subrange within any of the aforementioned ranges). In some non-limiting embodiments, the dose of each active agent may be about 60 mg. In some non- limiting embodiments, the dose of each active agent is about 1 mg. In some non-limiting embodiments, the dose of each active agent is about 2 mg.

[0089] In some non-limiting embodiments, the dose of each active agent is from 20 mg to 200 mg (e.g., from 20 mg to 175 mg, from 20 mg to 150 mg, from 20 mg to 100 mg, from 20 mg to 75 mg, from 30 mg to 175 mg, from 40 mg to 175 mg, from 45 mg to 175 mg, from 50 mg to 175 mg, from 30 mg to 150 mg, from 40 mg to 100 mg, from 45 mg to 75 mg, from 50 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; about 20 mg, about 30 mg, about 45 mg, or e.g., about 60 mg, or any subrange within any of the aforementioned ranges). In some non-limiting embodiments, the dose of each active agent is from about 12 mg to about 48 mg (e.g., from about 12 mg to about 42 mg, from about 12 mg to about 36 mg, from about 12 mg to about 30 mg, from about 18 mg to about 48 mg, from about 18 mg to about 42 mg, from about 24 mg to about 42 mg, from about 27 mg to about 42 mg, from about 24 mg to about 36 mg, from about 27 mg to about 33 mg, from about 28 mg to about 32 mg; e.g., about 24 mg,  about 27 mg, about 30 mg, about 33 mg, or about 36 mg, or any subrange within any of the aforementioned ranges).

[0090] In some non-limiting embodiments, the dosing regimen comprises administration of a dose of each active agent in a range of from 100 mg to 750 mg (e.g., from 100 mg to 725 mg, from 100 mg to 700 mg, from 100 mg to 675 mg, from 100 mg to 650 mg, from 100 mg to 625 mg, from 100 mg to 600 mg, from 100 mg to 575 mg, from 100 mg to 550 mg, from 100 mg to 525 mg, from 100 mg to 500 mg, from 100 mg to 475 mg, from 100 mg to 450 mg, from 100 mg to 425 mg, from 100 mg to 400 mg, from 100 mg to 375 mg, from 100 mg to 350 mg, from 100 mg to 325 mg, from 100 mg to 300 mg, from 100 mg to 275 mg, from 100 mg to 250 mg, or from 100 mg to 225 mg, from 100 mg to 200 mg, from 100 mg to 175 mg, from 100 mg to 150 mg, or from 100 mg to 125 mg, or any subrange within any of the aforementioned ranges).

[0091] In some non-limiting embodiments, the dosing regimen comprises administration of a dose of each active agent in a range of from 200 mg to 750 mg (e.g., from 200 mg to 725 mg, from 200 mg to 700 mg, from 200 mg to 675 mg, from 200 mg to 650 mg, from 200 mg to 625 mg, from 200 mg to 600 mg, from 200 mg to 575 mg, from 200 mg to 550 mg, from 200 mg to 525 mg, from 200 mg to 500 mg, from 200 mg to 475 mg, from 200 mg to 450 mg, from 200 mg to 425 mg, from 200 mg to 400 mg, from 200 mg to 375 mg, from 200 mg to 350 mg, from 200 mg to 325 mg, from 200 mg to 300 mg, from 200 mg to 275 mg, from 200 mg to 250 mg, or from 200 mg to 225 mg, or any subrange within any of the aforementioned ranges).

[0092] In some non-limiting embodiments, the dosing regimen comprises administration of a dose of each active agent in a range of from 300 mg to 750 mg (e.g., from 300 mg to 725 mg, from 300 mg to 700 mg, from 300 mg to 675 mg, from 300 mg to 650 mg, from 300 mg to 625 mg, from 300 mg to 600 mg, from 300 mg to 575 mg, from 300 mg to 550 mg, from 300 mg to 525 mg, from 300 mg to 500 mg, from 300 mg to 475 mg, from 300 mg to 450 mg, from 300 mg to 425 mg, from 300 mg to 400 mg, from 300 mg to 375 mg, from 300 mg to 350 mg, or from 300 mg to 325 mg, or any subrange within any of the aforementioned ranges).

[0093] In some non-limiting embodiments, the dosing regimen comprises administration of a dose of each active agent in a range of from 400 mg to 750 mg (e.g., from 400 mg to 725 mg, from 400 mg to 700 mg, from 400 mg to 675 mg, from 400 mg to 650 mg, from 400 mg to 625 mg, from 400 mg to 600 mg, from 400 mg to 575 mg, from 400 mg to 550 mg, from 400 mg  to 525 mg, from 400 mg to 500 mg, from 400 mg to 475 mg, from 400 mg to 450 mg, or from 400 mg to 425 mg, or any subrange within any of the aforementioned ranges).

[0094] In some non-limiting embodiments, the dosing regimen comprises administration of a dose of each active agent in a range of from 500 mg to 750 mg (e.g., from 500 mg to 725 mg, from 500 mg to 700 mg, from 500 mg to 675 mg, from 500 mg to 650 mg, from 500 mg to 625 mg, from 500 mg to 600 mg, from 500 mg to 575 mg, from 500 mg to 550 mg, or from 500 mg to 525 mg, or any subrange within any of the aforementioned ranges).

[0095] In some non-limiting embodiments, the dosing regimen comprises administration of a dose of each active agent in a range of from 600 mg to 750 mg (e.g., from 600 mg to 725 mg, from 600 mg to 700 mg, from 600 mg to 675 mg, from 600 mg to 650 mg, from 600 mg to 625 mg, or any subrange within any of the aforementioned ranges).

[0096] In some non-limiting embodiments, each active agent is provided in a concentration of about 1 nM, about 5 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 500 nM, about 550 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 ^M, about 2 ^M, about 3 ^M, about 4 ^M, about 5 ^M, about 6 ^M, about 7 ^M, about 8 ^M, about 9 ^M, about 10 ^M, about 15 ^M, about 20 ^M, about 25 ^M, about 30 ^M, about 35 ^M, about 40 ^M, about 45 ^M, about 50 ^M, about 60 ^M, about 70 ^M, about 75 ^M, about 80 ^M, about 90 ^M, about 100 ^M, about 125 ^M, about 150 ^M, about 175 ^M, about 200 ^M, about 250 ^M, about 300 ^M, about 350 ^M, about 400 ^M, about 500 ^M, about 600 ^M, about 700 ^M, about 750 ^M, about 800 ^M, about 900 ^M, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 250 mM, about 300 mM, about 400 mM, about 500mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, about 10 M, about 15 M, about 20 M, about 25 M, about 30 M, about 35 M, about 40 M, about 45 M, about 50 M, about 75 M, about 100 M,  or any range in between any two of the aforementioned concentrations, including said two concentrations as endpoints of the range, or any number in between any two of the aforementioned concentrations.

[0097] When administered orally, each dose may be protected from digestion. This can be accomplished either by complexing the dose with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the dose in an appropriately resistant carrier such as (but not limited to) a liposome, e.g., such as shown in U.S. Patent No.5,391,377.

[0098] In certain embodiments, the active agents of the dose can be administered within one hour of each other, within two hours of each other, within 3 hours of each other, within 6 hours of each other, within 12 hours of each other, within 24 hours of each other, within 36 hours of each other, within 48 hours of each other, within 72 hours of each other, or more. Thus, an individual who receives such treatment can benefit from a combined effect of the different therapeutic compounds.

[0099] The active agents of the present disclosure can be administered to a subject by any of a number of effective routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compounds may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Oral formulations may be formulated such that the active agents pass through a portion of the digestive system before being released, for example it may not be released until reaching the small intestine, or the colon. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0100] Tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active agent therein using, for example,  hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active agent can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0101] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0102] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0103] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0104] Formulations of the pharmaceutical compositions for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

[0105] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are  known in the art to be appropriate. In certain embodiments, the active agents of the present disclosure can be formulated into suppositories, slow-release formulations, or intrauterine delivery devices (IUDs).

[0106] Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.

[0107] Alternatively, or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0108] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active agents may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required. The ointments, pastes, creams and gels may contain, in addition to an active agent(s), excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to an active agent(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Such dosage forms can be made by dissolving or dispersing the active agent(s) in the proper medium. Absorption enhancers can also be used to increase the flux of the active agent(s) across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active agent(s) compound in a polymer matrix or gel.

[0109] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the present disclosure. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697 and 2005 / 004074 and U.S. Pat. No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to that of lacrimal fluids, aqueous humor or vitreous humor or are compatible with such fluids. A particular (but not limiting) route of administration is local administration (e.g., topical administration, such as eye drops, or administration via an implant).

[0110] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by  injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0111] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. For topical transdermal administration, the active agents are formulated into ointments, creams, salves, powders, and gels. Transdermal delivery systems can also include (for example but not by way of limitation) patches. The present compositions can also be administered in sustained delivery or sustained release mechanisms. For example, biodegradable microspheres or capsules or other biodegradable polymer configurations capable of sustained delivery can be included herein.

[0112] The compositions of the present disclosure may be formulated as implants, in the form of either biodegradable microparticles or small squared films comprising the microparticles of the present disclosure. The following describes methods of making such implants. Microparticles (e.g., 5-100 micrometers) containing different loadings of the compounds of the present disclosure, such as heteroarotinoids, are prepared by spray drying suspensions of the nanocrystals of the compounds and a biodegradable polymer (for example, polylactic acid of molecular weights 50,000 -100,000) or polylactic-co-glycolic acid copolymer (e.g., proportions 75:25 or 50:50). Microparticles may contain, e.g., 10 - 50% wt / wt drug:polymer and can be implanted alone, or in a biodegradable film, e.g., as an implantable chitosan-egg phosphatidylcholine (ePC) films. To make such ePC films, chitosan flakes and ePC can be dissolved in a 1% acetic acid at a ratio of 1:0.8 (wt / wt). Microparticles containing the drug in nanocrystal form are dispersed in the chitosan-ePC solution in different proportions (e.g., 1:3, 1:5, 1:7 and 1:10 wt / wt) to achieve the release of different drug doses. The resulting microparticle- chitosan-ePC suspension can be poured into a Teflon dish to have a 2-3 mm thickness and allowed to dry in a covered dessicator for 5 days. After the films are dry, they can be cut into small squares of 15x15 mm2. The implants can be made in different forms, including but not limited to thin films, rods, and wafers. Other biodegradable polymers that can be used to make implants include, but are not limited to, poly-lactic acid, poly-lactic-co- glycolic acid copolymer, poly-caprolactone, poly-sebacic acid, poly-adipic acid, poly(3-  hydroxybutyric acid), poly(3-hydroxybutyrate-co-3-hydroxyvalerate, poly-trimethylene carbonate, chitosan, chitin, gelatin, collagen, and hyaluronic acid.

[0113] In non-limiting embodiments, gels comprising the active agents of the present disclosure can be made by combining the active agents in various proportions to a sodium alginate gel base or carbomer jelly base to form a homogeneous gel suitable for topical application.

[0114] In non-limiting embodiments, ointments comprising the active agents of the present disclosure can be made by combining the active agents in various proportions to a Hydrophilic Petrolatum USP base, Lanolin, USP base or to Polyethylene glycol ointment, NF to form a homogeneous ointment suitable for topical application.

[0115] In non-limiting embodiments, creams comprising the active agents of the present disclosure can be made by combining the active agents in various proportions in suspension in water and glycerin (e.g., 20:1 parts) and emulsified in a mixture of e.g., Lanolin, Beeswax USP- NF and Cetyl alcohol, plus a Tween 80 and Span 80Several gel, ointment, and / or cream compositions that can be used are shown in García-Contreras L, Abu-Izza K, Lu DR. "Biodegradable cisplatin microspheres for direct brain injection: Preparation and characterization." Pharm Dev Technol (1997) 2(1): 53-65.

[0116] For inhalation, the present compositions can be delivered using any system known in the art, including (but not limited to) dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. For example (but not by way of limitation), the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another non-limiting aspect, the device for delivering the formulation to respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include (for example but not by way of limitation) air jet nebulizers.

[0117] For inhalation, the present compositions can be delivered using any system known in the art, including (but not limited to) dry powder aerosols, liquids delivery systems, air jet nebulizers, propellant systems, and the like. For example (but not by way of limitation), the pharmaceutical formulation can be administered in the form of an aerosol or mist. For aerosol administration, the formulation can be supplied in finely divided form along with a surfactant and propellant. In another non-limiting aspect, the device for delivering the formulation to  respiratory tissue is an inhaler in which the formulation vaporizes. Other liquid delivery systems include (for example, but not by way of limitation) air jet nebulizers.

[0118] Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0119] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the present disclosure include (but are not limited to) water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0120] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0121] In some cases, in order to prolong the effect of an active agent, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0122] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include  poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0123] As noted, effective amounts of the active agents may be administered orally, in the form of a solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions, solutions, elixirs or emulsions. Solid unit dosage forms can be capsules of the ordinary gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, and cornstarch, or the dosage forms can be sustained release preparations. The pharmaceutical composition may contain a solid carrier, such as a gelatin or an adjuvant. The tablet, capsule, and powder may contain from about .05 to about 95% of the active substance compound by dry weight. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol. When administered in liquid form, the pharmaceutical composition particularly contains from about 0.005 to about 95% by weight of the active agent(s). For example, a dose of about 10 mg to about 1000 mg once or twice a day could be administered orally.

[0124] In another embodiment, the active agents of the present disclosure can be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders, such as acacia, cornstarch, or gelatin, disintegrating agents such as potato starch or alginic acid, and a lubricant such as stearic acid or magnesium stearate. Liquid preparations are prepared by dissolving the active agents in an aqueous or non-aqueous pharmaceutically acceptable solvent which may also contain suspending agents, sweetening agents, flavoring agents, and preservative agents as are known in the art.

[0125] In one non-limiting aspect, the active agent(s) is incorporated in lipid monolayers or bilayers, such as (but not limited to) liposomes. Liposomes and liposomal formulations can be prepared according to standard methods and are also well known in the art, such as (but not limited to) those disclosed in U.S. Patent Nos. 6,110,490; 6,096,716; 5,283,185; 5,279,833; 4,235,871; 4,501,728; and 4,837,028.

[0126] In one non-limiting aspect, the compositions are prepared with carriers that will protect the active agent(s) against rapid elimination from the body, such as (but not limited to) a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as (but not limited to) ethylene vinyl  acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0127] The active agent(s) in general may be formulated to obtain compositions that include one or more pharmaceutically suitable excipients, surfactants, polyols, buffers, salts, amino acids, or additional ingredients, or some combination of these. This can be accomplished by known methods to prepare pharmaceutically useful dosages, whereby the active agent(s) is combined in a mixture with one or more pharmaceutically suitable excipients. Sterile phosphate- buffered saline is one non-limiting example of a pharmaceutically suitable excipient.

[0128] The acid addition salts may include, but are not limited to, 4-acetamidobenzoate, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate (besylate), benzoate, bisulfate, bitartrate, butyrate, calcium edetate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), cinnamate, citrate, cyclamate, digluconate, 2,5-dihydroxybenzoate, disuccinate, dodecyl sulfate (estolate), edetate (ethylenediaminetetraacetate), estolate (lauryl sulfate), ethane-1,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate, galactarate (mucate), gentisate (2,5- dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hexylresorcinate, hippurate, hydrabamine (N,N?- di(dehydroabietyl)-ethylenediamine), hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, isobutyrate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate (mesylate), methylsulfate, mucate, naphthalene-1,5-disulfonate (napadisylate), naphthalene-2-sulfonate (napsylate), nicotinate, nitrate, oleate, palmitate, p- aminobenzenesulfonate, p-aminosalicyclate, pamoate (embonate), pantothenate, pectinate, persulfate, phenylacetate, phenylethylbarbiturate, phosphate, polygalacturonate, propionate, p- toluenesulfonate (tosylate), pyroglutamate, pyruvate, salicylate, sebacate, stearate, subacetate, succinate, sulfamate, sulfate, tannate, tartrate, teoclate (8-chlorotheophyllinate), thiocyanate, triethiodide, undecanoate, undecylenate, and valerate.

[0129] Non-limiting examples of routes of administration of the compositions described herein include parenteral injection, e.g., by subcutaneous, intramuscular, or transdermal delivery. Other forms of injection include (but are not limited to) intravenous, intraarterial, intralymphatic, intrathecal, intraocular, intranasal, intracranial, intracerebral, intraperitoneal, or intracavitary injection. In parenteral administration, the compositions will be formulated in a unit dosage injectable form such as (but not limited to) a solution, suspension, or emulsion, in association with a pharmaceutically acceptable excipient. Such excipients are inherently nontoxic and nontherapeutic. Non-limiting examples of such excipients include saline, Ringer's  solution, dextrose solution, and Hanks' solution. Nonaqueous excipients such as (but not limited to) fixed oils and ethyl oleate may also be used. An alternative non-limiting excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as (but not limited to) substances that enhance isotonicity and chemical stability, including buffers and preservatives. The active agents can be delivered or administered alone or as pharmaceutical compositions by any means known in the art, such as (but not limited to) systemically, regionally, or locally, for example by intraarterial, intrathecal (IT), intravenous (IV), parenteral, intrapleural cavity, topical, oral, or local administration, as subcutaneous, intratracheal (e.g., by aerosol) or transmucosal administration (e.g., buccal, bladder, vaginal, uterine, rectal, and / or nasal mucosa). Administration can also be localized directly into a tumor. Administration into the systemic circulation by intravenous, inhalation, mucosal, or subcutaneous administration is typical. Intravenous administration can be, for example (but not by way of limitation), by infusion over a period such as (but not limited to) 30-90 min or by a single bolus injection, or by other regimens as described elsewhere herein.

[0130] For parenteral administration, for example, the active agent(s) may be dissolved in a physiologically acceptable pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable pharmaceutical carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative, or synthetic origin. The pharmaceutical carrier may also contain preservatives and buffers as are known in the art.

[0131] When an effective amount of the active agent(s) is administered by intravenous, cutaneous, or subcutaneous injection, the compound is particularly in the form of a pyrogen- free, parenterally acceptable aqueous solution or suspension. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is well within the skill in the art. A particular pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection may contain, in addition to the active agent(s), an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical compositions of the present disclosure may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

[0132] As noted, particular amounts and modes of administration can be determined by one skilled in the art of preparing formulations who can readily select the proper form and mode of administration, depending upon the particular characteristics of the active agent(s) selected, the condition to be treated, the stage of the condition, and other relevant circumstances using  formulation technology known in the art, described, for example, in Remington: The Science and Practice of Pharmacy, 22nd ed.

[0133] Additional pharmaceutical methods may be employed to control the duration of action of the active agents. Increased half-life and / or controlled release preparations may be achieved through the use of proteins or polymers to conjugate, complex with, and / or absorb the active agents as discussed previously herein. The controlled delivery and / or increased half- life may be achieved by selecting appropriate macromolecules (for example but not by way of limitation, polysaccharides, polyesters, polyamino acids, homopolymers, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, or carboxymethylcellulose, and acrylamides such as N-(2-hydroxypropyl) methacrylamide), and the appropriate concentration of macromolecules as well as the methods of incorporation, in order to control release.

[0134] Another possible method useful in controlling the duration of action of the active agents by controlled release preparations and half-life is incorporation of the active agents or their functional derivatives into particles of a polymeric material such as polyesters, polyamides, polyamino acids, hydrogels, poly(lactic acid), ethylene vinylacetate copolymers, copolymer micelles of, for example, polyethylene glycol (PEG) and poly(l-aspartamide). Additional pharmaceutical methods may be employed to increase bioavailability of the drug, such as Kolliphor HS15.

[0135] It is also possible to entrap the active agents in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatine-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules), or in macroemulsions. Such techniques are well known to persons having ordinary skill in the art.

[0136] When the active agents are to be used as an injectable material, they can be formulated into a conventional injectable carrier. Suitable carriers include biocompatible and pharmaceutically acceptable phosphate buffered saline solutions, which are particularly isotonic.

[0137] For reconstitution of a lyophilized product in accordance with the present disclosure, one may employ a sterile diluent, which may contain materials generally recognized for approximating physiological conditions and / or as required by governmental regulation. In this respect, the sterile diluent may contain a buffering agent to obtain a physiologically acceptable pH, such as sodium chloride, saline, phosphate-buffered saline, and / or other substances which are physiologically acceptable and / or safe for use. In general, the material for intravenous  injection in humans should conform to regulations established by the Food and Drug Administration, which are available to those in the field. The pharmaceutical composition may also be in the form of an aqueous solution containing many of the same substances as described above for the reconstitution of a lyophilized product.

[0138] The active agents can also be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, tauric acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as monoalkyl, dialkyl, trialkyl and aryl amines, and substituted ethanolamines.

[0139] In certain embodiments, the present disclosure includes an active agent composition wherein at least one of the active agents is coupled (e.g., by covalent bond) directly or indirectly to a carrier molecule.

[0140] Formulated compositions comprising the active agents of the present disclosure can be provided in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. Compositions can also take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents.

[0141] In some non-limiting methods, the patient is administered the active agent every one, two, three, or four weeks, for example. The dosage depends on the frequency of administration, condition of the patient, response to prior treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other factors.

[0142] The number of dosages administered may depends on the severity and temporal nature of the disorder (e.g., whether presenting acute or chronic symptoms) and the response of the disorder to the treatment. For acute disorders or acute exacerbations of a chronic disorder, between 1 and 10 doses may be used. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, the active agent may be administered at regular intervals, such as (but not limited to) weekly,  fortnightly, monthly, quarterly, every six months for at least 1, 5, or 10 years, or for the life of the patient.

[0143] Compositions comprising one or more of the active agents can be administered in a single dose treatment or in multiple dose treatments on a schedule and over a time period appropriate to the age, weight, and condition of the subject, the particular composition used, and the route of administration. In one non-limiting embodiment, a single dose of the composition according to the disclosure is administered. In other non-limiting embodiments, multiple doses are administered. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, degree of immunoprotection desired, or whether the composition is used for prophylactic or curative purposes. For example, in certain non-limiting embodiments, the composition is administered once per month, twice per month, three times per month, every other week, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, every other day, daily, twice a day, or three times a day. The duration of treatment (i.e., the period of time over which the composition is administered) can vary, depending on any of a variety of factors, e.g., subject response. For example (but not by way of limitation), the composition can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more.

[0144] The dosage of an administered active agent for humans will vary depending upon factors such as (but not limited to) the patient's age, weight, height, sex, general medical condition, and previous medical history. A dosage may be provided as several smaller amounts. For example, a single dosage of 500 mg may be administered as ten 50 mg tablets or capsules, or as five 100 mg tablets or capsules. The amounts of doses or dosages described herein may be provided in a single capsule, tablet, injection, infusion, or other more of delivery. Or, the amounts of drug which comprise the doses or dosages described herein may be provided in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) capsules, tablets, injections, infusions, or other modes of delivery.

[0145] In certain non-limiting embodiments, the recipient may be provided with a dosage of each active agent that is in the range of from about 1 mg to about 1000 mg. A lower or higher dosage also may be administered. In certain non-limiting embodiments, the dosage may be in the range of from about 25 mg to about 100 mg of the active agent per square meter (m2) of body  surface area for a typical adult, although a lower or higher dosage also may be administered. Dosages may be repeated as needed, for example (but not by way of limitation), once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. In certain non- limiting embodiments, the dosage can be provided as an infusion, for example as a single injection or as multiple injections. It may also be given less frequently, such as (but not limited to) every other week for several months, or more frequently, such as twice weekly, or by continuous infusion.

[0146] In some non-limiting embodiments, a per capsule or tablet dose may range from 25 mg to 200 mg (e.g., from 25 mg to 175 mg, from 25 mg to 150 mg, from 25 mg to 125 mg, from 25 mg to 100 mg, from 25 mg to 75 mg, from 25 mg to 70 mg, from 40 mg to 100 mg, from 40 mg to 75 mg, from 40 mg to 175 mg, from 40 mg to 150 mg, from 40 mg to 125 mg, from 40 mg to 70 mg, from 40 mg to 60 mg, or from 45 mg to 55 mg). In some non-limiting embodiments, the dose per capsule or tablet may be about 50 mg.

[0147] In some non-limiting embodiments, the dose per capsule or tablet is from 20 mg to 200 mg (e.g., from 20 mg to 175 mg, from 20 mg to 150 mg, from 20 mg to 100 mg, from 20 mg to 75 mg, from 30 mg to 175 mg, from 40 mg to 175 mg, from 45 mg to 175 mg, from 50 mg to 175 mg, from 30 mg to 150 mg, from 40 mg to 100 mg, from 45 mg to 75 mg, from 50 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; about 20 mg, about 30 mg, about 45 mg, or e.g., about 60 mg). In some non-limiting embodiments, the dose is from about 12 mg to about 48 mg (e.g., from about 12 mg to about 42 mg, from about 12 mg to about 36 mg, from about 12 mg to about 30 mg, from about 18 mg to about 48 mg, from about 18 mg to about 42 mg, from about 24 mg to about 42 mg, from about 27 mg to about 42 mg, from about 24 mg to about 36 mg, from about 27 mg to about 33 mg, from about 28 mg to about 32 mg; e.g., about 24 mg, about 27 mg, about 30 mg, about 33 mg, or about 36 mg).

[0148] In some non-limiting embodiments, the dosing regimen comprises administration of a loading dose, such as from 20 mg to 200 mg (e.g., from 20 mg to 175 mg, from 20 mg to 150 mg, from 20 mg to 100 mg, from 20 mg to 75 mg, from 30 mg to 175 mg, from 40 mg to 175 mg, from 45 mg to 175 mg, from 50 mg to 175 mg, from 30 mg to 150 mg, from 40 mg to 100 mg, from 45 mg to 75 mg, from 50 mg to 70 mg, from 55 mg to 65 mg, from 58 mg to 62 mg; e.g., about 60 mg). In some non-limiting embodiments, the dose is from about 12 mg to about 48 mg (e.g., from about 12 mg to about 42 mg, from about 12 mg to about 36 mg, from about 12 mg to about 30 mg, from about 18 mg to about 48 mg, from about 18 mg to about 42 mg, from about 24 mg to about 42 mg, from about 27 mg to about 42 mg, from about 24 mg to  about 36 mg, from about 27 mg to about 33 mg, from about 28 mg to about 32 mg; e.g., about 24 mg, about 27 mg, about 30 mg, about 33 mg, or about 36 mg).

[0149] In certain non-limiting embodiments, the per day dosage for administration to a subject is in a range of 1 mg / kg to 25 mg / kg, or 2 mg / kg to 24 mg / kg, or 3 mg / kg to 22 mg / kg, or 4 mg / kg to 20 mg / kg, or 5 mg / kg to 17.5 mg / kg, or 5 mg / kg to 15 mg / kg, or 5 mg / kg to 12.5 mg / kg, or 6 mg / kg to 25 mg / kg, or 6 mg / kg to 20 mg / kg, or 6 mg / kg to 15 mg / kg, or 6 mg / kg to 12 mg / kg.

[0150] In certain non-limiting embodiments, the amount of the active agent delivered to the subject per dose is in a range of about 100 mg to about 1000 mg, or about 200 mg to about 1000 mg, or about 300 mg to about 1000 mg, or about 400 mg to about 1000 mg, or about 250 mg to about 900 mg, or about 400 mg to about 900 mg, or about 400 mg to about 850 mg, or about 400 mg to about 800 mg, or about 400 mg to about 700 mg, or about 420 mg to about 840 mg, or about 500 mg to about 650 mg, or about 500 mg to about 1000 mg, or about 500 mg to about 900 mg, or about 500 mg to about 850 mg, or about 500 mg to about 800 mg, or about 500 mg to about 700 mg. The total dose to be delivered can be provided in a single capsule, tablet, injection, or bolus (or other dosage form), or in multiple capsules, tablets, injections, or boluses (or other dosage forms).

[0151] In at least certain non-limiting embodiments, when the active agent is provided in the form of a capsule or tablet, the capsule or tablet should disintegrate within about 5-10 minutes, and in the gastrointestinal (GI) tract the active agent should dissolve in about 30 minutes. In at least certain non-limiting embodiments, once the active agent is absorbed from the GI tract, it should produce a (therapeutic) concentration of at least 1.7 µg / ml, or at least 4 mM, in the target tissue. In at least certain non-limiting embodiments, the dosage form is designed to provide a therapeutic concentration of the active agent in the subject for at least about 12 to 24 hours, when the active agent is administered once a day.

[0152] Various embodiments of the present disclosure will be more readily understood by reference to the following examples and description, which as noted above are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to be limiting. The following detailed examples and methods which describe  various compositions of the present disclosure and are to be construed, as noted above, only as illustrative, and not limitations of the disclosure in any way whatsoever. EXAMPLES

[0153] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein after. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive. Example 1

[0154] Approximately one in four cervical cancer patients will have a recurrence of the cancer after it has removed with primary treatment. Even patients diagnosed with early-stage cervical cancer will experience cancer recurrence within 2-3 years after surgery at a rate of about 9 out of 100. In the United States, the median overall survival for recurrent and metastatic cervical cancer is approximately 16.5 months from the start of first line therapy despite compliance with national treatment guidelines. Furthermore, health care disparities worsen patient outcomes based on racial, ethnic and regional factors. Disparities in low-middle income countries result in cervical cancer being one of the top killers of their female populations.

[0155] Standard of care for cervical cancer consists of combinations of surgery, chemotherapy, and radiation, depending on disease severity. Patients presenting with advanced disease have the option of adding bevacizumab targeted at vascular endothelial growth factor or pembrolizumab targeted at programmed death-ligand 1 to their first line treatment regimens; however, there remains a need to improve patient prognosis. Given the high toxicities and morbidities caused by current therapies, targeting the elevated metabolism inherent in cancer is a current direction in new anti-cancer drug discovery and development strategies. This is particularly relevant for cervical cancer, because the vast majority of these tumors are caused by infection by HR-HPV. HR-HPV early oncoproteins cause metabolic reprogramming in cancer cells by interacting with cellular pathways to promote aerobic glycolysis. Alterations in glucose, lipid, and amino acid metabolism increase cervical cancer cell proliferation and survival in hostile microenvironments as they arise, metastasize, and respond to therapeutic treatments.

[0156] The SHetA2, currently in clinical trial for cervical and other cancers (clinicaltrial.gov: NCT04928508), has the potential to improve cervical cancer therapy because it inhibits and kills cervical cancer cells without causing toxicity by targeting HR-HPV  disrupted pathways. SHetA2 has been shown to cause mitochondrial damage and mitophagy in cervical cancer cells, indicating that it will disrupt mitochondrial metabolism. In endometrial cancer cells, SHetA2 inhibited both oxidative phosphorylation (OxPhos) and glycolysis, putting cells into quiescent states.

[0157] The molecular mechanisms of SHetA2 involve its binding to three HSP70s called mortalin, hsc70, and Grp78, and disrupting their folding and support of client proteins and protein complexes. In cervical cancer cell lines, SHetA2 disruption of hsc70 / AIF complexes is associated with accumulation of AIF and DNA damage in the nucleus and a mechanism of cell death that is caspase-independent and partially dependent upon mitophagy. Client proteins released from mortalin by SHetA2 treatment of endometrial cancer cells include metabolic enzymes aldehyde dehydrogenase 18 family member A1, cytidine triphosphate synthetase, malate dehydrogenase, and enoyl coenzyme A hydratase, short chain, 1. SHetA2 interference with mortalin is the likely mechanism by which it caused mitochondrial damage and inhibits OxPhos, because mortalin is primarily located in the mitochondria, where it serves a vital role in nuclear-encoded protein import and mitochondrial function. Since we have shown that cervical cancer cells exhibit a mechanism of SHetA2-induced cell death that is distinct from other cancer types, it is important to evaluate SHetA2 metabolic effects in cervical cancer cells in order to optimize therapeutic approaches for this specific cancer.

[0158] In the present example, we evaluated the metabolic and proteomic responses of cervical cancer cell lines to treatment with SHetA2, identified a metabolic-targeted strategy to increase SHetA2 sensitivity of these cell lines, and validated the results in vivo. METHODS Cell lines, culture conditions, and chemicals

[0159] HPV-positive and HPV-negative human cervical cancer cell lines including SiHa (# HTB-35™, positive for HPV 16), Ca Ski (#CRL-1550™, positive for HPV16), and C-33A (# HTB-31™, HPV negative) purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) were grown in mycoplasma-free condition and used within 20 passages. For cell culture studies, a 10 mM stock solution of SHetA2 was dissolved in DMSO (Neta Scientific, Hainesport, NJ, USA). Untreated control cultures were treated with the same final volume of DMSO administered to the treated cultures. For in vivo studies, SHetA2 synthesized in bulk by the US National Cancer Institute RAID Program was suspended in 30% Kolliphor HS 15 (Sigma Aldrich, Merck, Darmstadt, Germany). Untreated control animals were gavaged  with the same volume of 30% Kolliphor HS 15 given to the treated animals. 2-DG (#D8375, Sigma-Aldrich) and dissolved in water. Seahorse Metabolism assays

[0160] OxPhos and glycolysis metabolism were measured using the XF Cell Mito Stress kit and XF Glycolytic Rate Assay Kit, respectively, and the 96‐well XF Analyzer (all purchased from Agilent Bioscience, Santa Clara, CA, USA) according to the manufacturer’s protocol. Briefly, cells were grown on XF96 tissue culture plates overnight and then treated with 10 µM SHetA2 for 4 hours. One hour before the assay, the sensor cartridge was hydrated with 200 μL / well of supplied calibrant at 37°C without CO2. Then the plates were washed with the assay medium (DMEM, 200 μL, 3×) and incubated at 37 °C in a non-CO2incubator for 45 min. For the Mito Stress assay, oligomycin (1 µM), FCCP (2 µM) and a mixture of antimycin A and rotenone (0.5 µM each) were added into the appropriate ports of the cartridge and the instrument was calibrated. After calibration of the cartridge, the cell culture plate was loaded in the instrument. The OCR assay was run using the standard template.

[0161] In the glycolytic rate assay, initial steps were performed similar to the Mito Stress assay. A mixture of antimycin A and rotenone (0.5 µM each) and 2-DG (50 mM) were added into the appropriate cartridge ports. After cartridge calibration, the cell culture plate was washed again with assay medium and loaded in the instrument. The assay was run using the standard template. The Seahorse XF Glycolytic Rate Assay Report Generator was utilized to calculate ECAR and PER.

[0162] After the assays, media were aspirated, wells were washed once with PBS, and then proteins were extracted with M-PER (#78501, Thermofisher, Waltham, MA, USA) was added and protein concentrations measured using the BCA reagent. All data were normalized to total protein concentration. Mitochondrial Metabolic Viability Assay

[0163] Metabolic viability was measured using the MTT assay (#G4100, Promega Madison, WI, USA). Briefly, cells were cultured overnight in 96 well plates at densities of 4000 cells / well. Then the cells were treated with indicated concentration of drugs alone or in combinations for 72-hours followed by addition of MTT solution (15µl). After a 2-hour incubation in the presence of MTT solution, solubilizing or STOP solution was added and the culture plate was incubated overnight. The OD was measured at wavelengths of 570 nm and 620 nm using a BioTek Synergy H1 Micro Plate Reader and Gen52.09 Software. The average  ODs of triplicate treatments were normalized to the average ODs of the respective controls. GraphPad Prism 10 software was used to derive the IC50s, and efficacies (maximal percent growth inhibition activities). For each cell line, experiments were performed in triplicate and repeated 2-3 times with consistent results. Evaluation of Drug Combination Antagonistic, Additive versus Synergistic Interaction

[0164] Cells cultured for 24 h in 96-well plates were treated in triplicate with a two-fold dilution series of a 1:1 ratio of SHetA2 and 2-DG IC50 values. The fold effect of each drug treatment was determined with the MTT assay and calculated by dividing the average corrected OD of the treated cultures from the average corrected OD of control cultures treated with solvent only and then subtracting this quotient from the number 1. Average fold effects of each dose were used to draw isobolograms and calculate CI values at three ED levels that caused 50%, 75%, 90% and 95% cell reduction (ED50, ED75, ED90 and ED95, respectively) using CompuSyn Software, which is based on the Chou-Talalay Method. ATP production assay in 2-D and 3-D (Spheroid) Cultures

[0165] For 2-D cultures, 10,000 cells / well were grown into 96-well culture plates overnight to form monolayers, and then treated with varying concentrations of SHetA2, alone or in combination with 2-DG (0.25mM). After 24 hours, cell viability was measured CellTiter-glo® 2.0 Cell Viability Assay, which determines the number of metabolically active or viable cells by quantifying ATP.

[0166] For 3-D cultures, Ca Ski or SiHa cell lines were seeded (2000 cells / well) in 96-well clear round bottom ultra-low attachment microplates (Corning®, 7007) and incubated at 37°C in 5% CO2 for 48 hours to form spheroids. Subsequently, the spheroids were treated with varying concentrations of SHetA2 alone, or in combination with 2-DG. After 72 hours treatment, spheroid viability was assessed using CellTiter-Glo® 3D Cell Viability Assay (Promega, G9683) according to manufacturer’s instructions. The relative luminescence units were calculated by normalizing with respective controls treated with solvent only, and the data expressed as percentages. Fluorescent Immunocytochemical Staining and Imaging

[0167] Approximately 6 to 8000 SiHa or Ca Ski cervical cancer cells were seeded onto 8- chambered slides, and treated with SHetA2, 2-DG or both for 48 hours, followed by fixation with 4% paraformaldehyde, permeabilization with 0.1% TritonX‐100 and blocking with 4% BSA. Then, the cells were incubated with and MitoTracker™ Red CMXRos (#M7512, Thermo  Fisher Scientific, Waltham, MS, USA) for 45 minutes followed by primary antibody for γH2AX (#2577, Cell signaling, 1:100 dilutions) overnight, and then with Alexa Fluor 488‐ labeled secondary antibody for 1 hour. Afterward, DAPI was used to stain the nucleus and images were acquired with a 63X objective using a Zeiss Axio Observer Z1 (Göttingen, Germany). Mass spectrometry proteomic analysis

[0168] SiHa or C-33A cervical cancer cells were treated with 10 µM of SHetA2 or DMSO only for 24 h in triplicate and protein isolates were collected with M-PER. Samples were digested according to the filter aided sample preparation protocol and evaluated by liquid Chromatography with tandem mass spectrometry as described previously

[0022] . Murine Xenograft Tumor Drug Interaction Study

[0169] The animal study was approved by the OUHSC IACUC (Protocol #19-009-CHI). Four-week-old female athymic mice (Hsd:Athymic Nude-Foxn1numice) were purchased from ENVIGO, housed in temperature, humidity and light controlled conditions, acclimatized for 2 weeks, and then subcutaneously injected with 1.2 × 107SiHa cells using a biosafety hood. Tumor sizes were measured thrice / week, while body weights were determined weekly. Once tumors achieved ~50 mm3average volume ([width2× length] / 2), mice were randomized into 4 groups (N=8, determined by power analysis using previous data of SHetA2 effects on tumor size, mice that did not develop tumors were excluded) with no significant differences in tumor volume between groups (ANOVA, p > 0.05). Placebo or SHetA2 at 60 mg / kg were administered daily for 31 days by oral gavage.2-DG was given in drinking water. In the drug combination groups, SHetA2 and 2-DG were administered as they were when given as individual drugs. At the end of the study, animals were euthanized, and tumors and organs were collected and weighed. Histochemical and Immunohistochemical Analysis of Organs and Tumors

[0170] Liver, kidney, spleen, and tumors tissue were formalin-fixed and paraffin-embedded (FFPE). Five-micron sections were cut from the FFPE blocks, placed onto positively charged slides. For immunohistochemistry, slides were deparaffinized and rehydrated in an automated Multistainer (Leica ST5020, Leica, Buffalo Grove, IL, USA) followed by antigen retrieval using Leica Bond-IIITM, and blocking with 5% goat serum (01-6201, ThermoFisher Scientific, Waltham, MS, USA) for 30 min. Then, sections were incubated with peroxidase-blocking  reagent, followed by primary antibody for phosphorylated histone γH2AX (1:50 dilution, # 9718, Cell Signaling) 60 min, and then a poly-horse radish peroxidase secondary antibody. The 3, 3′-diaminobenzidine tetrahydrochloride was used as chromogen to detect staining and sections were counter-stained with hematoxylin, dehydrated (Leica ST5020) and mounted (Leica MM24). An antibody-specific positive control (non-experimental tissue) and a negative control (omission of primary antibody) were used to confirm antibody specificity. Digital micrographs were taken at the same magnification of a tumor-filled area of strong positive staining for each of six tumors in each treatment group, and the number of positively-stained nuclei scored using ImageJ software. Liver, kidney and spleen sections were cut, stained with hematoxylin and eosin and evaluated by experienced pathologist (Stan Lightfoot) for toxicity analysis. Statistical analysis

[0171] All experiments were independently repeated in triplicate at least twice. Data are expressed as mean ± SD for experimental replicates and SEM for biological replicates. The t‐ test or ANOVA were used to make comparisons between two groups or multiple groups, respectively. In situations where the data was not normally distributed, the Mann-Whitney test or Kruskal-Wallis test, were used for two groups or multiple groups, respectively. P <0.05 was considered statistically significant. Multiple paired t-test analysis was utilized to discover proteins that had q values (adjusted p values of < 0.05 / < 5% false discovery rate) in the triplicate results of protein levels in SHetA2 treated compared to untreated control cultures. Statistical analyses were done using GraphPad Prism 8 or 9 Software (GraphPad Software Inc., La Jolla, CA, USA). RESULTS Cervical cancer cells increase glycolysis in response to SHetA2 OxPhos inhibition

[0172] First, the metabolic responses to SHetA2 treatment of one HR-HPV negative (C-33 A) and two HR-HPV positive (Ca Ski and SiHa) cervical cancer cell lines was investigated in culture. In all three cell lines, a 4-hour treatment with 10 µM SHetA2 stopped OxPhos, as indicated by reduced OCR in Seahorse assays (FIG. 1(A-C)). FIG. 2 shows that SHetA2 reduced basal respiration (A), and completely blocked ATP production (B) and maximal respiration (C). FIG. 3 shows that SHetA2 also reduced spare respiratory capacity (A), which represents extra mitochondrial capacity to produce energy under stress or nutrient-deprived  conditions, and proton leak (B), which is an indicator of mitochondrial damage or a cellular response attempt to regulate mitochondrial ATP production.

[0173] Next, SHetA2 effects on glycolysis in these cell lines was evaluated by measuring ECAR in Seahorse assays. In contrast to the SHetA2 inhibition of OCR, FIG. 4 shows that SHetA2 caused increased ECAR in all three cell lines (A-C).

[0174] FIG. 5 shows that SHetA2 significantly increased basal PER (A), which includes both PER due to OxPhos and glycolysis; however, the effect of SHetA2 was mainly due to an increased contribution of glycolysis as indicated by GlycoPER (B). SHetA2 induction of glycolysis achieved equivalent or greater levels to the extra compensatory glycolysis induced by the combination rotenone and antimycin A treatment seen at 20 minutes on the x-axis. The level of SHetA2 induction of extra compensatory glycolysis above that of induced by rotenone and antimycin A was statistically significant in the HR-HPV negative, but not in the HR-HPV positive cell lines (C).

[0175] FIG.6 demonstrates that cervical cancer cells respond to SHetA2 interference with OxPhos by maximizing glycolysis. An energetic map of these data illustrates that SHetA2 decreased OxPhos and increased glycolysis in all three cell lines (A) with a maximum metabolic switch from OxPhos to glycolysis demonstrated in SiHa cells, compared to Ca Ski or C-33A. Evaluation of OxPhos OCR / glycolysis ECAR ratio (B) and the ratio of ATP produced by OxPhos to that produced by glycolysis (C) demonstrated significant inhibition of these ratios across all cell lines. Prevention of the glycolytic response increases SHetA2 sensitivity

[0176] To evaluate the role of compensatory glycolysis in SHetA2 resistance, medium that cannot fuel glycolysis was utilized. Results are shown in FIG. 7. Cells grown in glucose- medium can utilize both OxPhos and glycolysis to produce ATP, while cells grown in galactose-medium produce ATP predominantly through OxPhos. In the HR-HPV negative C- 33A SHetA2 sensitivity was similar in glucose and galactose media (A). The potencies of SHetA2 in the HR-HPV positive Ca Ski and SiHa cell lines were decreased in galactose compared to glucose media as indicated by the higher IC50 values in glucose compared to galactose media for these cell lines (B, C). The efficacy of SHetA2 was also increased in the SiHa cell line in galactose compared to glucose media as indicated by higher maximal SHetA2 reduction in percent viability in galactose compared to glucose media.

[0177] To translate these results toward clinical trial, the effects of inhibiting glycolysis with physiologically-achievable concentrations of 2-DG on SHetA2 sensitivities of the cell lines were measured. Results are shown in FIG. 8. Similar to results in galactose media,  treatment with 2-DG altered SHetA2 potencies in the HR-HPV positive Ca Ski and SiHa (B, C), but not in the HR-HPV negative C-33A (A).

[0178] Next, SHetA2 and 2-DG were tested to determine if they worked in an additive or a synergistic manner in reducing metabolic viability of the HR-HPV positive cell lines. As shown in FIG. 9 (A) the CI values derived for ED50, ED75 and ED90 were below the additive line and value of 1 indicating synergy in Ca Ski. Similar results were obtained for C-33A (not shown). For SiHa, all CI values indicated synergy, while strong synergy was indicated at ED95 as shown in FIG.10 (A). The DRI’s demonstrate that when the drugs are used in combination, a reduced amount of the drugs could be used to achieve the same efficacy as when the drugs are administered as single agents (Table 13). Mechanism of 2-DG / SHetA2 Synergy

[0179] To evaluate the mechanism of SHetA2 / 2-DG synergy, the mechanism of SHetA2 as single agent was first studied. SHetA2 has no effect on the cellular levels of its binding proteins, but instead releases client proteins from the SHetA2-binding proteins, often resulting in ubiquitin-mediated degradation and / or cellular re-localization of the client protein. Mass spectrometry was used to identify proteins present at significantly different levels in C-33 A and SiHa cultures treated for 24 hours with 10 µM SHetA2- versus vehicle. C-33 A cells responded by upregulating 15, and reducing 3, proteins (Table 14). Six of these 18 (33%) proteins are involved in mRNA translation (ribosomal proteins L4, 7A, L10L and S2; VARS1; and PUF60). SHetA2 reduced GOT2, which catalyzes the reversible interconversion of oxaloacetate and glutamate into aspartate and α-ketoglutarate in the malate-aspartate shuttle, and increased pyruvate carboxylase, which processes the pyruvate end product of glycolysis into oxaloacetate, which feeds into OxPhos. Only one of these 18 proteins, is involved in DNA repair, hepatoma-derived growth factor, which promotes homologous recombination repair of DNA double stranded breaks.1.tnt0h gneht l<gidls ort s htiseniLlleC5r9 8 3, ndecD 2.nE 03.m0siogg0 ra 1e> suaetnnCnlyaden a ehasc; tai0v9 33ee .737..cw0im-dsi gurr sD0 0 3n ino dCul E.0,1> ga hcni aVmf tnaeno Isi o arit C57 2 4grs gofa D 4.04.0 eneulnor)Ini E ysavtsRbem to aI D(r C01-xeC0e5 3 2dodn 3.d 3niG D 5. 5. m a,,nD-E 0 0 ;y25t moitd8.i0vi sti c0i noud 6na 59080 -9 7 37. dd geaatr2..8. 5.0neAtD15 8 4,saseE 1 mstnoe; dHiSg se3.3 ehte0h9 2tseD2.387.946.2r71 e.nrp -5 e4y ts e r4. alr uolf a E t0 1ucseV hguI5i 1.,lal3 2 3 8l s1-ms;0i cnotaRvD7D0.69.32.65.3099 .o .0gadeI E0C -f5o tnsud8.eana05 2 4 5 1 0u oletslaI D4.42.30.51.3yagvarR EreIesdawDnCo.ysa mer31 ne oliet2 2t,a)m;a54wtfbg aauTrniAtGAGcidsig.1-osDbeD- teD- n i re 02simH2H2 hoS S 1<ny.s1 TCfo ,.)sgl enomrsim.gl nsinure aiC Cka uSHliatsSvIyorgoatg darteCevnanahtotntnsetaRyrevocsiDeslaF%5>htiw2AteHSybseniLlleCrecnaClacivreCaHiSdnaA33Cn i rg7 7 9 1 8 8 7 7 9 4dpxdlo na52 2364641551555 3 699057403256 6 264383er E Fh .C1 -.1.1.1.1.1.1.1 -.1.1.1.1.1.1 -.1.1etlAsn e c rii ot5etmoa ec2rtnirenoc get b ialni A e 0t af niea 10s 1 4aL7torme 2 monmoes7050gn 6 alyLnniLnr N aP e ole. asiabinhoi apt vit1nkcm)e d 10kies nig 8n1ah d 9cn gal 12inyhnini dnriotxic ob eteot ioerto4nexni n1Go-m i aslyc ies a e oe th s 4p2lasAF tani nait oia mu tianit raemo rpopb)afrgac rpelkilprla pllzbecias isosortartma nata orttp biaP pe HG(D mstirn rocekis zsia rtenoeoc delocelUc(ylniceitaavmomaomoTnEul o rhPTJyli Hohto maenununoplp ursososog p aDG aAnDmca prfos itie tsy slypbirbirbirl1ob 2 P 2mT 5I AGA2JLF 1C819 A7050 0LTD0yO PT1 1 2 6FC 1 4L A7SG A F A GOR N DS IR CMPUPUP LPLPAD H K KDKNUNPPRR Re gnm ito isdoebiARNRt61100000000.0.00<636035052812.1.5515650630309312631 -64 -3658 21 34 901ee7 4 4 0 9 3 487 4MrT1 261 4 1 6 5 131 1 2 1 73f l630o or117145 1479 6 9 94 295 9 0650n23atn 1 866 19674259 822 07166eMoC2182 8514585665591 57191423299135 e6.3r17d.p l gn7396 46727050 97322078 172315 1xEoF ah 4.1C 1.1 -0.21.1 7-.3 1-.1 -0.41.1 3-.1 2-.1 -1.31.1 3-.1 -e ra6-2Ssa 2te n0 3 Xn 2gn g Ae otna g1 3L2S43nieeth mtaoit oie it snoa niitNRLsatavi hpolo Lnn niSah- OesarnN pir3apyls encsrci ellcatavuno it3 d ecniets Dar e ash tc ot mieietet niepla-'2reapsaepyoh tor or ortode n AfsaA 1 ena otmN Goz rt cpae nfr / ecf a etceredi ohsart 2p plpl rpkn ielNnarsRt- er Asiteo soa rpidmy tlppta cildni a a alailcuRtlt cias NcimP- Aopepniheps oiet momomomnJysthtyob liyrla anvE b DlehQTIGNtRorophrpe oPrpsosbosirbooss Feirbir obimr21lSSob 3F2 3;A PMSMAAU 6UD A 2P 01 332X4 A 3JPRRAmTVy NGUUPS B DQ EPT WL LPSPSI P_P P P PP PCNSTFL_R RL R R S

[0180] SiHa cells responded to SHetA2 by reducing eight, and upregulating three, proteins (Table 14). Five of these 11 (46%) proteins are involved in mRNA translation (ribosomal proteins L10a, L36, S23, S4 X-linked, and FtsJ3, involved in ribosome biogenesis). Four out of the 11 (28%) proteins (BTF3, DNA replication helicase / nuclease 2, protein phosphatase 2 phosphatase activator, and synuclein alpha) are directly or indirectly involved in DNA damage repair. There were no proteins significantly modulated by SHetA2 in common between the two cell lines.

[0181] To determine if these SHetA2-altered proteins are known to bind the SHetA2 target proteins, Ingenuity Pathway was used, which demonstrated that the majority of the SHetA2-altered proteins in each cell line are directly bound by Grp78, hsc70, and mortalin. These SHetA2-binding proteins and SHetA2-modulated proteins in cervical cancer cell lines connect through an integrated network. In summary, proteins altered by SHetA2 in cervical cancer cells act down-stream of the SHetA2-binding proteins, Grp78, hsc70, and mortalin, and are predominantly involved in protein synthesis in both cell lines and DNA damage repair.

[0182] To validate that 2-DG cooperates with SHetA2 through a metabolic mechanism, we demonstrated that inhibition of ATP production by SHetA2 was augment by co-treatment with 2- DG over a range of doses in both cell lines grown in 2D cultures (FIG.9 (B), FIG.10 (B)). Next, we demonstrated that 2-DG augmented SHetA2 inhibition of ATP production in the cell lines when they were cultured as 3D spheroids, which in comparison to 2D cultures, better represents the cell-cell interactions and exposures to nutrients that occur in vivo (FIG.9 (C), FIG.10 (C)).

[0183] Next, it was evaluated if 2-DG enhanced SHetA2 induction of cervical cancer cell death. Previously we demonstrated that the mechanism of SHetA2-induced cervical cancer cell death is caspase-independent, and involves AIF release from mitochondria and translocation to the nucleus where it promotes DNA damage. In the present work, the question of whether or not 2-DG enhances SHetA2-induced DNA damage was evaluated by imaging staining of cells with an antibody that recognizes the phosphorylated form of H2AX, a well-established marker of DNA double-strand breaks. Quantification of the imaging showed that 2-DG did not affect SHetA2- induction of nuclear γH2AX staining in either the Ca Ski cell line (FIGS.11 and 13) or the SiHa cell line (FIGS.12 and 14).  In vivo Validation of Drug Combination Efficacy and Mechanism

[0184] To validate the efficacy and mechanism of the 2-DG and SHetA2 combination observed in cell culture and begin to translate the results toward clinical application, it was investigated if inhibition of compensatory glycolysis by 2-DG could enhance in vivo efficacy of SHetA2 against SiHa tumor xenografts. Results are shown in FIG.15. When compared to each drug administered as a single agent or solvent only untreated controls, the SHetA2 and 2-DG drug combination significantly reduced tumor volume (A) and final tumor weight at the end of the experiment (B). There were no significant differences in the average body weights of the different treatment and untreated control groups throughout the treatment (C). There was no evidence of drug toxicity to the liver, kidney, or spleen histology (FIG. 16) or weights (Table 15). Quantification of nuclear γH2AX staining of the tumors in the various treatment groups confirmed that 2-DG did not significantly increase SHetA2-induced DNA damage (FIG.17). Table 15. Organ to Body Weight Ratios of Animals in Different Treatment Groups Treatment Group Organs Control SHetA22-DG SHetA2+ 2-DG 60 mg / kg500 mg / kgcombinationp valueLiver 0.050 ± 0.0050.047 ± 0.007 0.053 ± 0.001 0.055 ± 0.002Kidney 0.016 ± 0.0010.015 ± 0.001 0.016 ± 0.001 0.015 ± 0.001 0.601Spleen 0.010 ± 0.0020.0006 ± 0.001 0.008 ± 0.001 0.012 ± 0.002 0.086Discussion:

[0185] Despite cervical cancer being preventable via vaccine, it is very far from being eradicated, due to low vaccination rates against the HR-HPV causative factor, cancer recurrence, and drug resistance. Previously, it was demonstrated that SHetA2 inhibits HR-HPV-regulated pathways and causes a distinct mechanism of cell death in cervical cancer that is caspase- independent, whereas the mechanisms in other cancer types involve caspases. It is plausible that the HR-HPV in cervical cancer is responsible for this distinguishing mechanism of cell death, and could also drive distinguishing metabolic responses to various therapies

[0030] . Here it was demonstrated that cervical cancer cell lines exhibit reduced OxPhos and increased glycolysis in  response to treatment with SHetA2. This cervical cancer response is different from the reduction of both OxPhos and glycolysis observed in endometrial cancer cell lines.

[0186] The direct molecular mechanism of SHetA2 involves binding to the Grp78, hsc70, and mortalin chaperones, leading to disruption of their complexes with, and protection of, client proteins. In this Example, the majority of proteins identified to be present at significantly different levels in SHetA2-treated compared to control cells are known to be bound by these SHetA2 targeted chaperones and involved in regulation of protein synthesis and DNA damage repair. Reduction of OxPhos is a logical consequence of SHetA2’s interference with mortalin and mitochondrial function, leading to mitophagy and relocation of AIF from the mitochondria to the nucleus, where it promotes DNA damage. Yet interference with mitophagy and / or AIF expression only partially reduced SHetA2-induced cervical cancer cell death, indicating the contribution of other factors to the mechanism of cell death. In this Example, it has been demonstrated that elevated glycolysis can compensate for SHetA2’s inhibition of ATP production and tumor growth.

[0187] Although upregulation of glycolysis can compensate for reduced OxPhos ATP production, it does so at a lower efficiency. In this Example, it has been demonstrated that the SHetA2-induced glycolysis serves a compensatory function in the two HR-HPV positive cervical cancer cell lines, Ca Ski and SiHa, but not in the HR-HPV negative cell line C-33A. Because C- 33A has the highest sensitivity to SHetA2 in comparison to SiHa and Ca Ski, the ability of SHetA2 to kill the cell line may already be maxed out.

[0188] While not wishing to be bound by any particular theory, there are also several molecular mechanisms that could explain why C-33A is not sensitized to SHetA2 by glycolysis, including its HR-HPV and p53 statuses and proteomic response to SHetA2 treatment. A metabolomic study found that the HR-HPV positive SiHa and HeLa cell lines were more glycolytic in comparison to HR-HPV negative C-33A cancerous and HCKIT non-cancerous cell lines, and that C-33A had elevated arginine metabolism. Also, the R273C p53 mutation in C-33A may contribute to the lack of sensitization through its rewiring of metabolism. The similar R273H p53 mutation and other hot-spot p53 missense mutations have been shown to decrease OxPhos and increase glycolysis in breast cancer cells. In this current Example, the inability of glycolysis inhibition to sensitize the C-33A cell line to SHetA2 also may be related to the SHetA2 inhibition of GOT2 protein levels that was observed in C-33A. GOT2 is the intra-mitochondrial form of GOT that is important for bringing NADH produced by glycolysis into the mitochondria for regeneration into NAD+. The  SHetA2 reduction of GOT2 in C-33A may be caused by SHetA2 releasing GOT2 from hsc70 protection from degradation. GOT2 folding and import into the mitochondria has been shown to be regulated by a protein recognized by the N27F3-4 antibody that binds to both HSP70 and hsc70. Previously it was demonstrated that SHetA2 can disrupt hsc70 / AIF complexes, and hsc70 was identified as a relevant target for treatment of cervical cancer via association of its overexpression with cervical cancer patient survival probability and inhibition of AIF. All of these factors may contribute to C-33A’s greater sensitivity to SHetA2 and reduced reliance on glycolysis compared to the HR-HPV positive cervical cancer cell lines, Ca Ski and SiHa, thereby over-riding the ability of glycolysis inhibition to sensitize C-33A to SHetA2.

[0189] In this Example, the ability of glycolysis to compensate for SHetA2 inhibition of ATP production was demonstrated in HR-HPV positive cervical cancer cell lines using two methods. The first demonstrated that the cells were more sensitive to SHetA2 when cultured in galactose media, which supports OxPhos and not glycolysis, in comparison to when they were grown in glucose media, which supports both OxPhos and glycolysis. The second demonstrated that inhibition of glycolysis with 2-DG enhanced SHetA2 reduction of mitochondrial metabolic viability and production of ATP in cell culture. The enhanced inhibition of mitochondrial metabolic viability is likely due to 2-DG reduction of the glycolysis product pyruvate feeding mitochondrial metabolism. The mechanisms of 2-DG enhancement of SHetA2 inhibition of HR- HPV positive cervical cancer cell line culture and xenograft tumor growth were not associated with enhancement of SHetA2 induction of DNA damage.

[0190] The ongoing SHetA2 Phase 1 clinical trial (clinicaltrials.gov: NCT04928508) and extensive clinical use of 2-DG offer a pathway for rapid translation to SHetA2-based drug combination clinical trials. A Phase 1 trial of oral 2-DG in combination with docetaxel chemotherapy in patients with advanced solid tumors identified a safe starting dose. Since SHetA2 is also orally bioavailable, patients treated with a combination of SHetA2 and 2-DG could take their drugs at home. Concerns for potential toxicity caused by SHetA2 damage to mitochondria are reduced by the established SHetA2-resistance of mitochondria in non-cancer cells and lack of 12-week SHetA2 treatment effects on heart function. Overall SHetA2 caused no skin irritancy, mutagenicity, carcinogenicity, or any observable toxicity at doses 50-fold above the predicted therapeutic dose. Thus, combination of SHetA2 with 2-DG, or other specific glycolytic inhibitors  represents a rationale combination therapy that could be more effective than single SHetA2 treatment and without added toxicity.

[0191] The present Example thus demonstrates that elevated glycolysis partially compensates for reduced OxPhos in SHetA2-treated HR-HPV positive cervical cancer cell cultures. The compensatory glycolysis can be counteracted with culture media that does not support glycolysis or with 2-DG co-treatment in vitro. These results were validated in an in vivo xenograft model, supporting translation of combination treatment of SHetA2 and other Hets described herein with 2-DG and other inhibitors of glycolysis. Example 2

[0192] Tumor cells exhibit enhanced glycolytic / metabolic activity compared to normal cells, which is known as the “Warburg effect.” This allows them to sustain higher proliferative rates and resist some cell death signals. Subsequently, recent studies have provided supporting evidence that inhibition of glycolysis may exert preferential killing effect on cancer cells with compromised mitochondrial function or under hypoxia. Several natural and other molecules targeting various components of glycolysis have been evaluated.

[0193] 2-DG: Acting as a D-glucose mimic, 2-DG inhibits glycolysis due to the formation and intracellular accumulation of 2-DG6P, inhibiting the function of hexokinase and glucose-6- phosphate isomerase, and inducing cell death.2-DG has been widely studied in preclinical studies. Various animal and clinical studies demonstrate that 2-DG is safe and relatively non-toxic in animals and humans. A clinical trial suggests that 2-DG at doses up to 250 mg / kg appears safe for use in combination with radiation therapy in patients with glioblastoma multiforme. The most common adverse events after a 63 mg / kg dose of 2-DG were not life threating and included fatigue, sweating, dizziness, and nausea, mimicking the symptoms of hypoglycemia. Highly glycolytic cervical cancers largely resist chemoradiotherapy. Inhibition of glycolysis via 2-DG and intracellular redox metabolism sensitized highly glycolytic and radioresistant cervical cancers. Further study demonstrated that 2-DG increased the in vitro and in vivo responsiveness of cervical cancer cells to lapatinib. Also, 2-DG increased cisplatin resistance in human cervical cancer cells via up-regulating DDHs expression.

[0194] SHetA2 is an orally bioavailable drug, and patients treated with the combination of SHetA2 and 2-DG can take their drugs at home. Thus, the combination of SHetA2 with 2-DG,  both of which cause minimal toxicity, represents a rationale combination therapy that is more effective than single SHetA2 treatment and without added toxicity.

[0195] In this Example, the combined efficacy of SHetA2 and 2-DG in patients derived xenograft animal model of cervical cancer is studied.

[0196] SHetA2 is already in phase 1 human clinical trial for gynecological malignancy. In our preclinical studies, the investigational drug, SHetA2, inhibited cervical cancer xenografts growth without toxicity. We demonstrated that SHetA2 targets cervical cancer metabolism by disrupting mitochondrial dynamics and function, thereby inhibiting OxPhos and metabolic viability. However, these cells responded by upregulating glycolysis as a survival / resistance mechanism. Therefore, we hypothesized that inhibition of compensatory glycolysis will enhance the therapeutic efficacy of SHetA2 without toxicity in cervical cancer.

[0197] This Example provides a way to improve the efficacy of SHetA2 by using 2-DG. As shown herein, inhibition of glycolysis via 2-DG increased SHetA2 sensitivity of cervical cancer cell cultures and in a cervical cancer xenograft animal model. Since metabolic reprogramming leading to increased glycolysis has been shown to define malignant behaviors, chemoresistance, and cancer stem cells mediated recurrence, it represents a rational target for developing alternative therapies for cancer. Also, 2-DG has been shown to enhance the therapeutic efficacy of radiation, chemotherapies, and molecularly-targeted drugs in preclinical studies, and has been safely used in diagnostic imaging and in a Phase 1 trial in combination with docetaxel.

[0198] In particular, in a phase I trial in advanced solid tumors, daily oral 2-DG at 63 mg / kg in combination with docetaxel was chosen as the recommended Phase 2 dose. QTc prolongation was observed in 22% of the patients, and the authors speculated that this was related to reversible hyperglycemia that occurred in 100% of the patients at the 63 to 88 mg dose levels. There was no pharmacokinetic interaction between 2-DG and docetaxel. Efficacy was not evaluated in the single-agent 2-DG trial; however, of the 34 patients in the combination trial, eleven (32%) had stable disease, one (3%) had a partial response, and twenty-two (66%) had progressive disease.

[0199] Since 2-DG has an already well-established safety profile, these evidences support the feasibility of a drug combination approach to inhibit tumor progression and recurrence. The drug combination of the present disclosure provides a more effective, less toxic alternative to current standard of care, thereby improving the survival and quality of life of women with cervical cancers which are highly glycolytic in nature or with similar other cancers. The reduced or no adverse  effects could also reduce the cost of care and impact on family members and care-givers for these patients.

[0200] While the present disclosure has been described in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the present disclosure be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the present disclosure. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments only and are presented in the cause of providing what is believed to be the most useful and readily understood description of procedures as well as of the principles and conceptual aspects of the presently disclosed methods and compositions. Changes may be made in the formulation of the various compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the present disclosure.

[0201] In at least certain aspects, the present disclosure is directed to the following non-limiting illustrative embodiments:

[0202] Illustrative embodiment 1. A pharmaceutical composition, comprising: at least one heteroarotinoid and at least one glycolytic inhibitor disposed in a pharmaceutically acceptable excipient or vehicle, wherein administration of the pharmaceutical composition yields a synergistic therapeutic effect.

[0203] Illustrative embodiment 2. The pharmaceutical composition of Illustrative embodiment 1, wherein the synergistic therapeutic effect is a synergistic anti-cancer effect.

[0204] Illustrative embodiment 3. The pharmaceutical composition of Illustrative embodiments 1 or 2, wherein the at least one glycolytic inhibitor is selected from the group consisting of 2- deoxy-D-glucose (2-DG), 2-fluoro-2-deoxy-D-glucose (2-FG), 2-chloro-2-deoxy-D-glucose (2- CG), 2-bromo-2-deoxy-D-glucose (2-BG), acetylated derivatives of 2-DG (e.g., WP1208, WP1124, WP1125, WP1209, WP1210, WP1122, and WP1211); 3-bromopyruvate (3-BP); 1-(4- pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2- propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6-aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-  6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations thereof.

[0205] Illustrative embodiment 4. The pharmaceutical composition of Illustrative embodiments 1 - 3, wherein the at least one heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, NHet90, and combinations thereof.

[0206] Illustrative embodiment 5. The pharmaceutical composition of any one of Illustrative embodiments 1-4 selected from a Het-drug combination represented in Tables 10-12.

[0207] Illustrative embodiment 6. The pharmaceutical composition of any of Illustrative embodiments 1-5, wherein the at least one heteroarotinoid comprises a dosage in a range of from about 1 mg / kg to about 100 mg / kg, and the at least one glycolytic inhibitor comprises a dosage in a range of from about 1 mg / kg to about 100 mg / kg.

[0208] Illustrative embodiment 7. The pharmaceutical composition of any of Illustrative embodiments 1-6, wherein the at least one heteroarotinoid and the at least one glycolytic inhibitor comprise a weight-to-weight ratio in a range of from about 40,000:1 to about 1:40,000.

[0209] Illustrative embodiment 8. A kit or commercial package, comprising: at least one heteroarotinoid, and at least one glycolytic inhibitor configured for conjoint administration, wherein the conjoint administration of the pharmaceutical composition yields a synergistic therapeutic effect.

[0210] Illustrative embodiment 9. The kit or commercial package of Illustrative embodiment 8, wherein the synergistic therapeutic effect is a synergistic anti-cancer effect.

[0211] Illustrative embodiment 10. The kit or commercial package of Illustrative embodiments 8 or 9, wherein the at least one glycolytic inhibitor is selected from the group consisting of 2- deoxy-D-glucose (2-DG), 2-fluoro-2-deoxy-D-glucose (2-FG), 2-chloro-2-deoxy-D-glucose (2- CG), 2-bromo-2-deoxy-D-glucose (2-BG), acetylated derivatives of 2-DG (e.g., WP1208, WP1124, WP1125, WP1209, WP1210, WP1122, and WP1211); 3-bromopyruvate (3-BP); 1-(4- pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2- propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6-aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)- 6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations thereof.

[0212] Illustrative embodiment 11. The kit or commercial package of any one of Illustrative embodiments 8-11, wherein the at least one heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, and NHet90.

[0213] Illustrative embodiment 12. The kit or commercial package of any one of Illustrative embodiments 8-11 comprising a Het-drug combination represented in Tables 10-12.

[0214] Illustrative embodiment 13. A method for treating a subject in need of such therapy, comprising the step of administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one heteroarotinoid and at least one glycolytic inhibitor; wherein administration of the pharmaceutical composition yields a synergistic therapeutic effect.

[0215] Illustrative embodiment 14. The method of Illustrative embodiment 13, further defined as a cancer treatment method that results in at least one of a reduction of tumor size, a reduction of tumor growth, a stabilization of disease (inhibition of tumor growth), an inhibition of cancer metastases, a cancer cell death, and an inhibition of tumor recurrence, wherein the synergistic therapeutic effect is a synergistic anti-cancer effect.

[0216] Illustrative embodiment 15. The method of Illustrative embodiments 14, wherein the cancer comprises cervical squamous cell carcinoma, endocervical adenocarcinoma, ovarian serous cystadenocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, lung squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, esophageal carcinoma, stomach adenocarcinoma, uterine carcinosarcoma, uterine corpus endometrial carcinoma, liver hepatocellular carcinoma, kidney renal papillary cell carcinoma cholangiocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, mesothelioma, ovarian cancer, anal cancer, vulvar cancer, penile cancer, or vaginal cancer.

[0217] Illustrative embodiment 16. The method of any of Illustrative embodiments 13-15, wherein the at least one glycolytic inhibitor is selected from the group consisting of 2-deoxy-D- glucose (2-DG), 2-fluoro-2-deoxy-D-glucose (2-FG), 2-chloro-2-deoxy-D-glucose (2-CG), 2- bromo-2-deoxy-D-glucose (2-BG), acetylated derivatives of 2-DG (e.g., WP1208, WP1124, WP1125, WP1209, WP1210, WP1122, and WP1211); 3-bromopyruvate (3-BP); 1-(4-pyridinyl)- 3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-  one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6-aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)- 6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations thereof.

[0218] Illustrative embodiment 17. The method of any of Illustrative embodiments 13-16, wherein the at least one heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, and NHet90.

[0219] Illustrative embodiment 18. The method of any one of Illustrative embodiments 13-17, wherein the pharmaceutical composition comprises a Het-drug combination represented in Tables 10-12.

[0220] Illustrative embodiment 19. The method of any of Illustrative embodiments 13-18, wherein the at least one heteroarotinoid is administered in a dose range of from about 1 mg / kg to about 100 mg / kg, and the at least one is administered in a dose range of from about 1 mg / kg to about 100 mg / kg.

[0221] Illustrative embodiment 20. The method of any of Illustrative embodiments 13-19, wherein the at least one heteroarotinoid and the at least one glycolytic inhibitor are administered in a weight-to-weight ratio in a range of from about 40,000:1 to about 1:40,000.

[0222] Illustrative embodiment 21. A method for treating a subject in need of such therapy, comprising the step of conjointly administering to the subject a therapeutically effective amount of at least one heteroarotinoid and a therapeutically effective amount of at least one glycolytic inhibitor; wherein the conjoint administration of the at least one heteroarotinoid and the at least one glycolytic inhibitor yields a synergistic therapeutic effect.

[0223] Illustrative embodiment 22. The method of Illustrative embodiment 21, further defined as a cancer treatment method that results in at least one of reduction of tumor size, reduction of tumor growth, stabilization of disease (inhibition of tumor growth), inhibition of cancer metastases, cancer cell death, and inhibition of tumor recurrence, wherein the synergistic therapeutic effect is a synergistic anti-cancer effect.

[0224] Illustrative embodiment 23. The method of Illustrative embodiment 22, wherein the cancer comprises cervical squamous cell carcinoma, endocervical adenocarcinoma, ovarian serous cystadenocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, lung squamous  cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, esophageal carcinoma, stomach adenocarcinoma, uterine carcinosarcoma, uterine corpus endometrial carcinoma, liver hepatocellular carcinoma, kidney renal papillary cell carcinoma cholangiocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, mesothelioma, ovarian cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer.

[0225] Illustrative embodiment 24. The method of any of Illustrative embodiments 21-23, wherein the at least one glycolytic inhibitor is selected from the group consisting of 2-deoxy-D- glucose (2-DG), 2-fluoro-2-deoxy-D-glucose (2-FG), 2-chloro-2-deoxy-D-glucose (2-CG), 2- bromo-2-deoxy-D-glucose (2-BG), acetylated derivatives of 2-DG (e.g., WP1208, WP1124, WP1125, WP1209, WP1210, WP1122, and WP1211); 3-bromopyruvate (3-BP); 1-(4-pyridinyl)- 3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1- one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6-aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)- 6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations of the above.

[0226] Illustrative embodiment 25. The method of any of Illustrative embodiments 21-24, wherein the at least one heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, and NHet90.

[0227] Illustrative embodiment 26. The method of any one of Illustrative embodiments 21-25, comprising a Het-drug combination represented in Tables 10-12.

[0228] Illustrative embodiment 27. The method of any of Illustrative embodiments 21-26, wherein the at least one heteroarotinoid is administered in a dose range of from about 1 mg / kg to about 100 mg / kg, and the at least one glycolytic inhibitor is administered in a dose range of from about 1 mg / kg to about 100 mg / kg.

[0229] Illustrative embodiment 28. The method of any of Illustrative embodiments 21-27, wherein the at least one heteroarotinoid and the at least one glycolytic inhibitor are administered in a weight-to-weight ratio in a range of from about 40,000:1 to about 1:40,000.

[0230] Illustrative embodiment 29. The method of any of Illustrative embodiments 21-28, wherein the at least one heteroarotinoid is administered prior to the at least one glycolytic inhibitor.

[0231] Illustrative embodiment 30. The method of any of Illustrative embodiments 21-28, wherein the at least one glycolytic inhibitor is administered prior to the at least one heteroarotinoid.

[0232] Illustrative embodiment 31. The method of any of Illustrative embodiments 21-28, wherein the at least one glycolytic inhibitor is administered simultaneously with the at least one heteroarotinoid.

[0233] Illustrative embodiment 32. The method of any of Illustrative embodiments 21-31, wherein the at least one glycolytic inhibitor sensitizes the cancer to the at least one heteroarotinoid.

[0234] Illustrative embodiment 33. The method of Illustrative embodiment 21-32, wherein the at least one glycolytic inhibitor functions to mitigate an increase in glycolysis caused by the administration of the at least one heteroarotinoid.

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Claims

What is claimed is:

1. A pharmaceutical composition, comprising: at least one heteroarotinoid; at least one glycolytic inhibitor; and a pharmaceutically acceptable excipient or vehicle.

2. The pharmaceutical composition of claim 1, wherein the heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, NHet90, and combinations thereof.

3. The pharmaceutical composition of claim 1, wherein the glycolytic inhibitor is selected from the group consisting of 2-deoxy-D-glucose (2-DG); 2-fluoro-2-deoxy-D-glucose (2-FG); 2- chloro-2-deoxy-D-glucose (2-CG); 2-bromo-2-deoxy-D-glucose (2-BG); acetylated derivatives of 2-DG; 3-bromopyruvate (3-BP); 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6-aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations of the above.

4. The pharmaceutical composition of claim 1, comprising a Het-drug combination represented in Tables 10-12.

5. The pharmaceutical composition of claim 1, wherein the heteroarotinoid comprises a dosage in a range of from about 1 mg / kg to about 100 mg / kg, and the glycolytic inhibitor comprises a dosage in a range of from about 1 mg / kg to about 100 mg / kg.

6. The pharmaceutical composition of claim 1, wherein the heteroarotinoid and glycolytic inhibitor comprise a weight-to-weight ratio in a range of from about 40,000:1 to about 1:40,000.

7. A kit or commercial package, comprising: at least one heteroarotinoid; and  at least one glycolytic inhibitor for conjoint administration with the heteroarotinoid.

8. The kit or commercial package of claim 7, wherein the heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, and NHet90.

9. The kit or commercial package of claim 7, wherein the glycolytic inhibitor is selected from the group consisting of 2-deoxy-D-glucose (2-DG); 2-fluoro-2-deoxy-D-glucose (2-FG); 2-chloro- 2-deoxy-D-glucose (2-CG); 2-bromo-2-deoxy-D-glucose (2-BG); acetylated derivatives of 2-DG; 3-bromopyruvate (3-BP); 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6- aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations of the above.

10. The kit or commercial package of claim 7, comprising a Het-drug combination represented in Tables 10-12.

11. A method for treating a subject in need of such therapy, comprising the step of: administering a therapeutically effective amount of a pharmaceutical composition comprising at least one heteroarotinoid compound and at least one glycolytic inhibitor to the subject; wherein administration of the pharmaceutical composition yields a synergistic effect compared to an effect of the heteroarotinoid alone and an effect of the glycolytic inhibitor alone.

12. The method of claim 11, further defined as a cancer treatment method that results in at least one of reduction of tumor size, reduction of tumor growth, stabilization of disease (inhibition of tumor growth), inhibition of cancer metastases, cancer cell death, and inhibition of tumor recurrence.

13. The method of claim 11, wherein the heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, and NHet90.

14. The method of claim 11, wherein the glycolytic inhibitor is selected from the group consisting of 2-deoxy-D-glucose (2-DG); 2-fluoro-2-deoxy-D-glucose (2-FG); 2-chloro-2-deoxy- D-glucose (2-CG); 2-bromo-2-deoxy-D-glucose (2-BG); acetylated derivatives of 2-DG; 3- bromopyruvate (3-BP); 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6- aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations of the above.

15. The method of claim 12, wherein the cancer is at least one of cervical squamous cell carcinoma, endocervical adenocarcinoma, ovarian serous cystadenocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, lung squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, esophageal carcinoma, stomach adenocarcinoma, uterine carcinosarcoma, uterine corpus endometrial carcinoma, liver hepatocellular carcinoma, kidney renal papillary cell carcinoma cholangiocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, mesothelioma, ovarian cancer, anal cancer, vulvar cancer, penile cancer, and vaginal cancer.

16. The method of claim 11, wherein the pharmaceutical composition comprises a Het-drug combination represented in Tables 10-12.

17. The method of claim 11, wherein the heteroarotinoid is administered in a dose range of from about 1 mg / kg to about 100 mg / kg, and the second compound is administered in a dose range of from about 1 mg / kg to about 100 mg / kg.

18. The method of claim 11, wherein the heteroarotinoid and the glycolytic inhibitor are administered in a weight-to-weight ratio in a range of from about 40,000:1 to about 1:40,000.

19. A method for treating a subject in need of such therapy, comprising the step of: conjointly administering to the subject a therapeutically effective amount of at least one heteroarotinoid compound and a therapeutically effective amount of at least one glycolytic inhibitor; wherein administration of the at least one heteroarotinoid compound and the at least one glycolytic inhibitor yields a synergistic effect compared to an effect of the heteroarotinoid alone and an effect of the glycolytic inhibitor alone.

20. The method of claim 19, further defined as a cancer treatment method that results in at least one of reduction of tumor size, reduction of tumor growth, stabilization of disease (inhibition of tumor growth), inhibition of cancer metastases, cancer cell death, and inhibition of tumor recurrence.

21. The method of claim 19, wherein the heteroarotinoid is selected from the group consisting of SHetA2, SHetA3, SHetA4, SHetC2, SHetD3, SHetD4, SHetD5, SHet50, SHet65, SHet100, OHet72, NHet17, NHet86, and NHet90.

22. The method of claim 19, wherein the glycolytic inhibitor is selected from the group consisting of 2-deoxy-D-glucose (2-DG); 2-fluoro-2-deoxy-D-glucose (2-FG); 2-chloro-2-deoxy- D-glucose (2-CG); 2-bromo-2-deoxy-D-glucose (2-BG); acetylated derivatives of 2-DG; 3- bromopyruvate (3-BP); 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one) (PFK15); (2E)-3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one) (3PO); dichloroacetate (DCA); lonidamine (LND); 6- aminonicotinamide (6AN); benitrobenrazide (BNBZ); 1,4,5-oxathiazandioxide-4,4 (GP-2250, misetionamide); (22E,24R)-6β-methoxyergosta-7,9(11),22-triene-3β,5α-diol (MOTD); and metformin (MET), and combinations of the above.

23. The method of claim 20, wherein the cancer is at least one of cervical squamous cell carcinoma, endocervical adenocarcinoma, ovarian serous cystadenocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, lung squamous cell carcinoma, kidney renal clear cell carcinoma, lung adenocarcinoma, head and neck squamous cell carcinoma, pancreatic  adenocarcinoma, esophageal carcinoma, stomach adenocarcinoma, uterine carcinosarcoma, uterine corpus endometrial carcinoma, liver hepatocellular carcinoma, kidney renal papillary cell carcinoma cholangiocarcinoma, bladder urothelial carcinoma, breast invasive carcinoma, mesothelioma, ovarian cancer, anal cancer, vulvar cancer, penile cancer, and vaginal cancer.

24. The method of claim 19, wherein the at least one heteroarotinoid compound and the at least one glycolytic inhibitor comprises a Het-drug combination represented in Tables 10-12.

25. The method of claim 19, wherein the heteroarotinoid is administered in a dose range of from about 1 mg / kg to about 100 mg / kg, and the second compound is administered in a dose range of from about 1 mg / kg to about 100 mg / kg.

26. The method of claim 19, wherein the heteroarotinoid and the glycolytic inhibitor are administered in a weight-to-weight ratio in a range of from about 40,000:1 to about 1:40,000.

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