Methods and compositions for inhibiting GAPDH

Abstract

Methods of inhibiting GAPDH using certain oxathiazine-like and / or related compounds.

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for treating, suppressing, preventing, or alleviating disorders and diseases in a subject by administering one or more anti-GAPDH agents of the present disclosure.

Background Art

[0002] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in complex cellular pathways. In addition to the cytoplasm where most of GAPDH is located under basal conditions, GAPDH is also found in particulate fractions such as the nucleus, mitochondria, and vesicular fractions. When cells are exposed to various stress factors, dynamic intracellular redistribution of GAPDH occurs. In particular, GAPDH is an important enzyme for energy metabolism and the production of ATP and pyruvate by aerobic glycolysis in the cytoplasm. Increased gene expression and enzymatic function of GAPDH are associated with cell proliferation and tumor formation, but conditions such as oxidative stress impair the catalytic activity of GAPDH, leading to cell senescence and apoptosis. Various interaction partners of GAPDH, including proteins, various RNA species, and telomeric DNA, have been identified, but the mechanism underlying the effect of GAPDH on cell proliferation remains unclear.

[0003] Several studies have shown that GAPDH has multifaceted functions independent of its canonical role in glycolysis. The functional diversity of GAPDH is mainly due to post-translational modifications at various amino acid residues or protein-protein interactions that change its localization from the cytosol to the nucleus, mitochondria, or extracellular microenvironment. The non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair, and RNA nuclear export, and are observed in physiological and pathological conditions such as cancer and neurodegenerative disorders.

[0004] The oligomeric state of GAPDH and its tendency to aggregate mainly depend on various signaling molecules. The redox-sensitive cysteine residue of this enzyme, which contains Cys-152 in the active site, is also a target of reactive oxygen species (ROS) or reactive nitrogen species (RNS). As a result, GAPDH aggregation is affected by several other stimuli that induce cellular oxidative / nitrosative stress. Besides cancer, due to the functional diversity of this enzyme, it has been found that changes in GAPDH are involved in several other diseases, especially neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

[0005] The non-glycolytic roles of GAPDH include regulation of gene expression, DNA repair and replication, neurodegeneration, pathogenesis, tubular bundling, protein-protein interaction, RNA nuclear export, and pathophysiological functions such as apoptosis and autophagy. For example, it has been discovered that GAPDH acts as an important element of the Oct-1 co-activator complex in the transcriptional induction of the histone H2B gene during the S phase of the cell cycle. Interestingly, GAPDH directly interacts with Oct-1 and has a unique activation domain that can be associated with the basal transcriptional machinery.

[0006] GAPDH can act as an intracellular glucose sensor and also stimulate autophagic degradation. In fact, during glucose starvation, AMPK-dependent phosphorylation of GAPDH is essential for the activation of SIRT1 and the stimulation of autophagy. Under these conditions, cytoplasmic GAPDH is phosphorylated by activated AMPK, promoting the redistribution of GAPDH into the nucleus. In the nucleus, GAPDH directly interacts with SIRT1, displaces the repressor of SIRT1, and increases the SIRT1 deacetylase activity. Generally, multiple activities of GAPDH are related to its translocation to the nucleus or different intracellular compartments in addition to its well-characterized cytosolic localization in glycolysis.

[0007] Nuclear GAPDH is involved in various functions such as autophagy and cell death, DNA repair, and protection of telomeres from rapid degradation. The accumulation of GAPDH in the nucleus promotes the decline of its glycolytic activity. When DNA is damaged during oxidative stress, simultaneous nitrosylation and nuclear translocation of GAPDH occur, and GAPDH can bind to poly(ADP-ribose) polymerase 1 (PARP1) or directly to damaged DNA. Under these stress conditions, PARP1 is activated by damaged DNA and synthesizes poly(ADP-ribose) using NAD + . Furthermore, nuclear-translocated GAPDH binds to PARP1 and activates PARP1. The overactivation of PARP1 depletes intracellular NAD + , so that the NAD + -binding site of GAPDH is released, and the enzyme acquires the ability to bind to DNA. When there is a site cleaved into single-stranded DNA fragments, GAPDH forms a stable covalent adduct with this damage. Therefore, the formation of an irreversible complex between GAPDH and DNA seems to be a suicidal event that can prevent DNA repair and lead to cell death in case of the accumulation of several damages.

[0008] Furthermore, since the presence of nuclear GAPDH is involved in the initiation of one or more apoptotic cascades, a specific role of GAPDH in neuronal apoptosis has been shown. There are various case studies demonstrating the role of GAPDH in several neurodegenerative diseases such as HD and PD. An interesting hypothesis is that GAPDH binds to mutant proteins associated with these diseases, nuclear translocation occurs, and the presence of GAPDH there is involved in the initiation of apoptosis. Therefore, an increase in nuclear GAPDH has been reported in postmortem PD brains associated with degeneration-susceptible substantia nigra dopaminergic neurons. Furthermore, GAPDH has been recognized as a major component of amyloid plaques in the Alzheimer's disease brain and has also been reported to interact with neurodegenerative disease-related proteins including amyloid-β protein precursor (AβPP). Non-native GAPDH isoforms can bind to soluble Aβ species, indicating a direct involvement of GAPDH in amyloid aggregation.

[0009] Cytosolic GAPDH is also involved in apoptosis in a form that is mainly regulated by post-translational modifications and protein-protein interactions. In fact, GAPDH is phosphorylated by Akt2 at Thr237, which is close to the binding site of Siah1, preventing its binding to Siah1 and apoptosis. The formation of the GAPDH / Akt2 complex has been identified as a mechanism that favors the survival of tumor cells and avoids apoptosis in ovarian cancer cells. Another way in which cytosolic GAPDH is involved in tumor survival is the escape from caspase-independent cell death (CICD). By stabilizing Akt in its activated and phosphorylated form, overexpressed GAPDH prevents the nuclear translocation of FoxO, which regulates Bcl-6, an inhibitor of Bcl-xL with anti-apoptotic function.

[0010] Furthermore, the functional relevance between cytosolic GAPDH and microtubule dynamics, vesicular transport, and membrane mobilization and fusion has been demonstrated by many studies. GAPDH can interact with tubulin and actin under normal conditions and with stress fibers during stress, and its glycolytic function that promotes its inactivation is regulated. These roles in cell transport are regulated by post-translational phosphorylation of the enzyme and can be involved in the transport of the early secretory pathway. A serine / threonine kinase promoted by Rab2 acts as a regulator of GAPDH-mediated secretory activity and determines the direction of membrane transport. GAPDH also has a role as a chaperone containing labile heme in cells. GAPDH helps in the transport and delivery of many cytosolic hemes. GAPDH binds to exogenous and endogenous hemes and makes them available to downstream protein targets that can be in the cytosol (e.g., iNOS) or in the nucleus. In this way, GAPDH not only protects cells from heme toxicity but is also involved in mobilization.

[0011] Under basal conditions, the level of GAPDH in mitochondria is very low and strongly increases under stress conditions such as serum starvation and DNA damage. When GAPDH is endogenously expressed, mitochondrial GAPDH induces apoptosis-promoting mitochondrial membrane permeabilization (MMP) by binding to voltage-dependent anion channel 1 (VDAC1). Exogenous expression of mitochondria also causes loss of the inner membrane potential, swelling of the matrix, permeabilization of the inner mitochondrial membrane, and release of two apoptosis-promoting proteins such as cytochrome c and apoptosis-inducing factor (AIF). Furthermore, during myocardial ischemia and reperfusion (I / R), GAPDH is significantly associated with mitochondria and is found to promote the direct uptake of damaged mitochondria into multi-organellar lysosome-like (LL) structures for elimination, independent of the macroautophagy pathway.

[0012] The complex functions of this enzyme are related to its translocation to different intracellular compartments. GAPDH-mediated autophagy and GAPDH aggregation may affect the proliferation of cancer cells and neurodegenerative disorders. Cancer-related factors may modulate the nuclear translocation of GAPDH, which is essential for the regulation of autophagy and cell death mechanisms. Autophagy stimulation by nuclear GAPDH may affect the fate of cancer cells, act as a survival-promoting factor in cancer cells, and support the energy consumption brought about by rapid cell proliferation even under stress conditions. Furthermore, the formation of GAPDH aggregates or the interaction of GAPDH with specific disease-related proteins may be involved in neuronal cell death and mitochondrial dysfunction. Considering its diverse and complex functionality, effective therapeutic agents that safely and effectively modulate, inhibit, and regulate the activity of GAPDH will provide a powerful tool in a wide range of medical fields.

Summary of the Invention

Problems to be Solved by the Invention

[0013] Accordingly, there has long been an unmet need for new compositions and methods that not only treat, suppress, prevent, or alleviate disorders and diseases in a subject by administering one or more anti-GAPDH agents, but also improve the performance, outcome, and tolerability of existing therapeutic agents.

Means for Solving the Problems

[0014] In one aspect, the present disclosure includes a method of inhibiting GAPDH, comprising administering to a subject in need of GAPDH inhibition a compound that is hydrolyzed or metabolized in vivo to form hydroxymethylamide isethionate.

[0015] In one aspect, the present disclosure includes a method of inhibiting GAPDH in a subject in need thereof by administering to the subject a composition comprising a compound of the present disclosure.

[0016] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity in a subject's cells by about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% by administering to the subject a composition comprising a compound of the present disclosure.

[0017] In one aspect, the present disclosure includes a method of reducing or inhibiting the production of adenosine triphosphate (ATP) in a subject in need thereof by administering to the subject a composition comprising a compound of the present disclosure.

[0018] In one aspect, the present disclosure includes a method of preventing, suppressing, or alleviating at least one sign or symptom of a disease, disorder, or condition resulting from or associated with GAPDH activity in a subject in need thereof by administering to the subject a composition comprising a compound of the present disclosure.

[0019] In one aspect, the present disclosure includes a method of increasing the production or localization of reactive species in a tumor of a subject in need thereof by administering to the subject a composition comprising a compound of the present disclosure.

[0020] In one aspect, the present disclosure includes a method of preventing, suppressing, or reducing at least one side effect of a drug administered to a subject suffering from a GAPDH-mediated disease, disorder, or condition by administering to the subject a composition comprising a compound of the present disclosure.

[0021] In one aspect, the present disclosure includes identifying a test compound that inhibits GAPDH by combining a test compound and a solvent to form a solution, contacting the solution with recombinant GAPDH in a buffer to form a reaction mixture, subjecting an aliquot of the reaction mixture to an enzyme activity assay, detecting a change in NAD+ concentration in the enzyme activity assay, and identifying a test compound that reduces the NAD+ concentration in the enzyme activity assay compared to a control solvent.

[0022] In one aspect, the present disclosure includes obtaining a biological sample containing cells from a subject, lysing the cells, monitoring GAPDH activity in the lysed cells as a biomarker for a GAPDH-mediated disease, and administering to the subject a composition comprising a GAPDH inhibitor.

[0023] In one aspect, the present disclosure includes administering a GAPDH inhibitor compound to a subject, obtaining peripheral blood mononuclear cells (PBMCs) from the subject, lysing the PBMCs, monitoring the GAPDH activity in the lysed PBMCs, subjecting the lysed PBMCs to an enzyme activity assay, detecting a change in NAD+ concentration in the enzyme activity assay, monitoring the inhibition of GAPDH by the administered GAPDH inhibitor based on a decrease in NAD+ concentration in the enzyme activity assay compared to a control solvent, determining the degree of inhibition of GAPDH in the PBMCs, and identifying the subject as a candidate suitable for treatment with the GAPDH inhibitor compound when the degree of inhibition of GAPDH by the GAPDH inhibitor compound exceeds a predetermined threshold. A method for identifying a candidate suitable for treatment with a GAPDH inhibitor compound is included.

[0024] In one aspect, the present disclosure includes a treatment method that includes identifying a candidate suitable for treatment with a GAPDH inhibitor according to the method of claim 20 or 21 and treating the candidate with a compound of the present disclosure.

[0025] In one aspect, the present disclosure includes a method for treating macular degeneration in a subject in need thereof by administering to the subject a composition comprising a compound of the present disclosure.

[0026] In some aspects, the present disclosure may include a composition comprising isethionic acid hydroxymethylamide or a pharmaceutically acceptable salt, hydrate, ester or solvate thereof, and isethionic acid hydroxymethylamide or a pharmaceutically acceptable salt, hydrate, ester or solvate thereof and an excipient, buffer or carrier.

[0027] In one aspect, the present disclosure includes a complex or conjugate of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and a compound of the present disclosure.

[0028] Other features and characteristics of the subject matter of the present disclosure, as well as methods of operation, functions of related elements of the structure and combinations of components, and economies of manufacture, will become more apparent by consideration of the following description, drawings and appended claims, which are all part of this specification.

Brief Description of the Drawings

[0029]

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DETAILED DESCRIPTION OF THE INVENTION

[0030] Aspects of the subject matter of the present disclosure may be embodied in various forms, but the following description is merely intended to disclose some of these forms as specific examples of the subject matter encompassed by the present disclosure. Accordingly, the subject matter of the present disclosure is not intended to be limited to the forms or aspects so described and illustrated.

[0031] To facilitate understanding of the present invention, a number of terms are defined below. The terms defined herein have meanings commonly understood by those skilled in the art related to the present invention. Terms that do not specify a quantity (such as "a", "an", and "the") are not intended to refer only to a single entity, but include general classifications that can be used to illustrate specific examples. The technical terms in this specification are used to describe specific aspects of the present invention, but their use does not define the present invention, except as outlined in the claims.

[0032] The terms "inhibit", "reduce" or "prevent", or any variation of these terms, when used in the claims and / or this specification, include any measurable decrease or complete inhibition to achieve the desired result.

[0033] The anti-GAPDH agents of the present disclosure can be administered to any subject in need of inhibition of GAPDH activity. Such subjects are at risk of or may be suffering from various diseases, disorders and conditions. For example, such diseases, disorders and conditions may be characterized by glycolytic disorders, disorders of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reactions, immune responses, excessive angiogenesis, dysfunction of apoptosis in normal cells, and / or disorders of autophagy. As used herein, the expression "GAPDH-mediated disorder, disease or condition" encompasses any one or more disorders, diseases or conditions in a subject in need of inhibition of GAPDH activity, including, but not limited to, glycolytic disorders, disorders of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reactions, immune responses, excessive angiogenesis, dysfunction of apoptosis in normal cells, and / or disorders of autophagy, and includes, but is not limited to, any one or more disorders, diseases or conditions discussed herein.

[0034] The present disclosure provides methods and compositions for inhibiting GAPDH target cells in which aerobic glycolysis is observed. In this type of metabolism, only a small fraction of the glucose flux is used for energy production and can be decreased by GAPDH inhibition. Aerobic glycolysis is seen in almost all types of tumor cells but not in normal cells. Thus, the present disclosure provides methods and compositions having a broad range of anti-GAPDH activity without general toxicity to normal cells. Further, the present disclosure provides methods and compositions for modulating cells that operate by aerobic glycolytic energy metabolism, such as activated endothelial cells and activated immune cells.

[0035] In some embodiments, the present disclosure provides methods and compositions for irreversibly inhibiting GAPDH. Thus, the present disclosure provides surprising and unexpected advantages over existing therapies, such as antibodies, which require continuous administration and have limited efficacy. The present disclosure provides methods for permanently inactivating GAPDH by irreversibly binding to its active site.

[0036] In some embodiments, the present disclosure provides methods and compositions for modulating mitochondrial function and protein production to reduce, inhibit, prevent, and / or eliminate cancer stem cells (CSCs). In some embodiments, the present disclosure provides methods and compositions for increasing reactive species, such as reactive oxygen species, in tumors and cancerous cells, thereby reducing the viability of cancer cells without affecting normal cells. In some embodiments, the present disclosure provides methods and compositions for inducing the restoration of the normal extracellular matrix of the fibrotic tissue surrounding cancer cells / tumors. In some embodiments, the present disclosure provides methods and compositions for reducing, inhibiting, preventing, and / or removing cytokines. In some embodiments, the present disclosure provides methods and compositions for administering to a subject having a therapy / condition that results in cytokine release or an increase in cytokine levels. In some embodiments, the present disclosure provides methods and compositions for reducing, inhibiting, preventing, and / or removing cytokines in immunotherapies, including but not limited to T cell-induced therapies such as CAR-T and bispecific therapies, without interfering with cytotoxicity to target cancer cells.

[0037] In some aspects, the present disclosure relates to acralasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal & neuronal neuropathy:AMAN, Barlow's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), eosinophilic granulomatosis with polyangiitis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackievirus myocarditis, CREST syndrome (limited scleroderma), Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, graft-versus-host disease (GVHD), granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis, pemphigoid gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy,Neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Persistent-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, type II, type III, rheumatoid polymyalgia, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, carcinomas, leukemias, lymphomas, melanomas, myelomas, sarcomas, metastatic solid tumors and tumors including but not limited to mixed carcinomas, cancers, skin diseases (including but not limited to psoriasis, telangiectasia, wound granularization, scleroderma, angiogenesis as a result of infections (e.g., cat scratch disease, bacterial ulcers, etc.)), macular degeneration or age-related blindness, diabetic ulcers, chronic ulcers and wounds, stroke, traumatic brain injury, retinal angiogenesis, corneal angiogenesis (such as those caused by trachoma, infections, inflammation, transplantation or trauma), diabetic retinopathy, diabetic retinal edema, diabetic macular edema, ischemic retinopathy, hypertensive retinopathy, obstructive retinopathy, retinopathy of prematurity, post-traumatic angiogenesis, post-infection angiogenesis, post-transplantation angiogenesis, angiogenesis after retinal detachment or retinal degeneration, neovascular glaucoma, anterior chamber and / or angle neovascularization,Methods and compositions for treating, reducing, suppressing or preventing choroidal neovascularization (CNV), subretinal neovascularization, retrolental fibroplasia, ocular histoplasmosis syndrome, myopic degeneration, retinitis pigmentosa, uveitis, rubeosis, retrolental fibroplasia, ocular histoplasmosis and idiopathic central serous chorioretinopathy, amyotrophic lateral sclerosis, sarcoidosis, scleroderma, lupus, Parkinson's disease, sclerosis, Stevens-Johnson syndrome, neoplasms, von Willebrand disease, vasculitis, and Kawasaki disease are also provided.

[0038] The present disclosure also provides methods and compositions for treating subjects suffering from cardiovascular diseases including, but not limited to, atherosclerosis, restenosis, atherosclerotic plaques and hemangiomas. Atherosclerosis is a form of chronic vascular injury in which some properties of normal vascular smooth muscle cells (VSMCs) in the arterial wall change, generating a dense capillary network in atherosclerotic plaques. These fragile microvessels can cause bleeding, leading to blood clotting and subsequent reduction in blood flow to the myocardium and risk of heart attack. Restenosis typically occurs after coronary artery bypass surgery, endarterectomy and heart transplantation, particularly after balloon angioplasty, atherectomy, laser ablation or intravascular stent placement.

[0039] As used herein, the terms "substantially" and "substantial" refer to a significant degree or extent. For example, when used with an event, situation, characteristic or property, the term refers not only to the case where the event, situation, characteristic or property occurs exactly, but also to the case where the event, situation, characteristic or property occurs very nearly, such as to account for the typical levels of tolerance or variability of the examples described herein.

[0040] As used herein, the term "about" is used to give latitude to the endpoints of a numerical range by defining that a given value may be "slightly above" or "slightly below" the endpoints. The latitude of this term may depend on the particular variable and a determination based on experience and the relevant description in this specification is within the knowledge of those skilled in the art. For example, in one aspect, the latitude may be within about ±10% of the numerical value. In another aspect, the latitude may be within about ±5% of the numerical value. In a further aspect, the latitude may be within about ±2%, ±1% or ±0.05% of the numerical value.

[0041] Generally, as used herein, the term "or" includes "and" and "and / or".

[0042] As used herein, multiple compounds or steps may be presented in a common list for convenience. However, these lists should be interpreted as if each member of the list were individually identified as a separate and distinct member. Thus, unless otherwise indicated, the individual members of such lists should not be construed as being substantially equivalent to any other member of the same list solely based on the fact that they are presented in a common group.

[0043] The compounds of the present invention may be useful in the form of free acid, free base, pharmaceutically acceptable salts, pharmaceutically acceptable hydrates, pharmaceutically acceptable esters, pharmaceutically acceptable solvates, pharmaceutically acceptable prodrugs, pharmaceutically acceptable metabolites, and pharmaceutically acceptable stereoisomers. All of these forms are within the scope of the present invention. In practice, the use of these forms corresponds to the use of the neutral compound.

[0044] "Pharmaceutically acceptable salts", "hydrates", "esters" or "solvates" refer to salts, hydrates, esters or solvates of the compounds of the present invention that have the desired pharmacological activity and are not undesirable biologically or otherwise. Using organic acids, salts, hydrates, esters or solvates such as acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, p-toluenesulfonates, bisulfates, sulfamates, sulfates, naphthalenates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, oxalates, tosylates and undecanoates can be produced. Using inorganic acids, salts, hydrates, esters or solvates such as hydrochlorides, hydrobromides, hydroiodides and thiocyanates can be produced. Other pharmaceutically acceptable salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, phosphates, tartrates, fumarates, maleates, oxalates, acetates, propionates, succinates, mandelates, mesylates, besylates and tosylates.

[0045] Salts, hydrates, esters or solvates can also be formed with organic bases. Pharmaceutically acceptable base addition salts of acidic compounds can be formed with organic and inorganic bases by conventional methods. Examples include hydroxides, carbonates and bicarbonates of alkali metals and alkaline earth metals such as sodium hydroxide, potassium hydroxide, calcium hydroxide, potassium carbonate, sodium bicarbonate, magnesium carbonate, ammonia, primary, secondary and tertiary amines, etc. Also, the aluminum salt of this compound can be obtained by treating the corresponding sodium salt with a suitable aluminum complex such as aluminum chloride hexahydrate. Examples of non-toxic organic bases include, but are not limited to, triethylamine, butylamine, piperazine and tris(hydroxymethyl)-methylamine. Examples of suitable salts, hydrates, esters or solvates of bases include hydroxides, carbonates and bicarbonates of ammonia, alkali metal salts such as sodium salts, lithium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, and zinc salts. Organic bases suitable for the formation of pharmaceutically acceptable base addition salts, hydrates, esters or solvates of the compounds of the present invention include non-toxic and sufficiently strong organic bases to form such salts, hydrates, esters or solvates. For illustration, such organic bases include monoalkylamines, dialkylamines and trialkylamines such as methylamine, dimethylamine, triethylamine and dicyclohexylamine; monohydroxyalkylamines, dihydroxyalkylamines or trihydroxyalkylamines such as monoethanolamine, diethanolamine and triethanolamine; amino acids such as arginine and lysine; guanidine; N-methyl-glucosamine; N-methyl-glucamine; L-glutamine; N-methyl-piperazine; morpholine; ethylenediamine; N-benzyl-phenethylamine; (tris(hydroxy-methyl)aminoethane, etc. See, for example, "Pharmaceutical Salts," J. Pharm. Sci., 66:1, 1-19 (1977).Therefore, the basic nitrogen-containing group can be quaternized with an agent including lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; long-chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aralkyl halides such as benzyl and phenethyl bromides.

[0046] The salts, hydrates, esters, or solvates of the basic compound can be prepared by dissolving the free base of the oxathiazine-like compound in an aqueous solution or aqueous alcohol solution or other suitable solvent containing an appropriate acid or base, and isolating the salt by evaporating the solution. Alternatively, the free base of the oxathiazine-like compound may be reacted with an acid, and the oxathiazine-like compound having an acidic group may be reacted with a base. The reaction is carried out in an organic solvent. In this case, the salt can be separated directly or obtained by concentrating the solution.

[0047] "Pharmaceutically acceptable prodrug" refers to a derivative of the compound of the present invention that exhibits its pharmacological effect(s) after undergoing in vivo changes. Prodrugs are formulated for the purpose of improving chemical stability, patient acceptance and compliance, bioavailability, duration of action, organ selectivity, formulation (e.g., increasing hydrosolubility), and / or reducing side effects (e.g., toxicity). Prodrugs can be readily prepared from the compounds of the present invention using methods known in the art, such as those described in Burger's Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, 949-982 (1995). For example, the compounds of the present invention can be converted into prodrugs by converting one or more of the hydroxy or carboxy groups to esters. Furthermore, N-protected forms of the compounds of the present invention are also included as non-limiting examples of pharmaceutically acceptable prodrugs of the compounds of the present invention.

[0048] "Pharmaceutically acceptable metabolite" refers to a drug that has undergone metabolic conversion. Most drugs, after entering the body, can serve as substrates for chemical reactions that can change their physical properties and biological effects. These metabolic conversions usually affect the polarity of the compound and change the way the drug is distributed in and excreted from the body. In some cases, drug metabolism is required for therapeutic effect. For example, antineoplastic drugs in the group of antimetabolites need to be converted to the active form after being transported to cancer cells. Since most drugs undergo some kind of metabolic conversion, the biochemical reactions involved in drug metabolism can be diverse. The main site of drug metabolism is the liver, although other tissues may also be involved.

[0049] Furthermore, certain compositions, concentrations, dosing regimens, dosages, syndromes or pathologies, steps, etc. can be considered in relation to a particular aspect. This is merely for convenience, and it is understood that such disclosure applies equally to other aspects found in this specification. For example, the method steps, active agents, kits or lists of compositions described with respect to the method of administering an anti-GAPDH agent of the present disclosure treat, prevent, suppress or alleviate at least one sign or symptom of a disease, disorder or pathology resulting from or related to GAPDH activity; treat, prevent, suppress or alleviate at least one side effect of a drug administered to a subject suffering from a disease, disorder or pathology resulting from or related to GAPDH activity; treat, prevent, suppress or alleviate the occurrence of signs or symptoms of a disease, disorder or pathology resulting from or related to GAPDH activity; modulate angiogenesis; regulate angiogenesis; modulate neovascularization; and also directly support aspects related to method steps, active agents, kits or compositions that regulate GAPDH activity, even if these method steps, active agents, kits or compositions are not described again in relation to that aspect in this specification.

[0050] As used herein, the terms "treating" or "treatment", which are well understood in the art, mean an approach for obtaining a beneficial or desired result, including clinical results. Beneficial or desired clinical results include, without regard to detectability or undetectability, alleviation or improvement of one or more symptoms or conditions, reduction in the extent of a disease, stabilization of a disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of a disease state, reduction in the recurrence of a disease, and remission (partial or complete), but are not limited thereto. "Treating" and "treatment" may also mean prolonging survival as compared to the expected survival in the absence of treatment. The methods described herein may be useful for preventing or prophylaxis of a disease in addition to being useful as a method of treatment. As used herein, the term "treating" may refer to any administration of a compound of the invention, including (i) preventing or suppressing a disease in a mammal, e.g., a human, experiencing or manifesting the pathological condition or overall symptoms of the disease (i.e., halting further progression of the pathological condition and / or overall symptoms), or (ii) ameliorating a disease in a mammal, e.g., a human, experiencing or manifesting the pathological condition or overall symptoms of the disease (i.e., reversing the pathological condition and / or overall symptoms). The term "controlling" includes preventing, treating, eradicating, ameliorating, or otherwise reducing the severity of the condition being controlled.

[0051] Concentrations, amounts, and other numerical data may be expressed or presented in a range format in this specification. Such range format is used merely for convenience and brevity, and it should be understood that it is to be interpreted flexibly to include not only the numerically explicitly listed values as the limits of the range, but also all of the individual values or sub-ranges subsumed within that range, as if each numerical value and sub-range were explicitly listed. By way of example, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly listed values of about 0.01 to about 2.0, but also the individual values and sub-ranges within the specified range. For this reason, individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5 are included in this numerical range. Further, such interpretation should apply regardless of the breadth of the range or the characteristics being described. In addition, note that unless otherwise specified, all percentages are weight percentages.

[0052] In understanding the scope of the present disclosure, the term "including" or "comprising" and its derivatives, as used herein, are intended to be open-ended terms that identify the presence of the recited features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unrecited features, elements, components, groups, integers, and / or steps. The above also applies to terms having similar meanings such as the terms "including", "having", and their derivatives. The term "consisting" and its derivatives, as used herein, are intended to be closed terms that identify the presence of the recited features, elements, components, groups, integers, and / or steps, but exclude the presence of other unrecited features, elements, components, groups, integers, and / or steps. The term "consisting essentially of", as used herein, is intended to identify the presence of the recited features, elements, components, groups, integers, and / or steps, as well as the presence of those that do not substantially affect the basic novel characteristics (which may be plural) of the features, elements, components, groups, integers, and / or steps. It is understood that any reference to any one of these transitional terms (i.e., "including", "consisting", or "consisting essentially of") directly supports a substitution to any other transitional term that is not specifically used. For example, the modification of the term from "including" to "consisting essentially of" is directly supported by this definition.

[0053] Several oxathiazine-like compounds are described in International Application PCT / IB2015 / 059741, filed December 17, 2015, which is hereby incorporated by reference in its entirety. In certain embodiments, an oxathiazine-like compound of formula I is used according to the present invention, wherein R is H, a linker or group cleavable in vivo, or a group that detaches in an aqueous solution, and R1 and R2 are independently H, alkyl, aryl, substituted alkyl, substituted phenyl, substituted aryl, or combinations thereof. In some embodiments, the substituted alkyl, substituted phenyl, or substituted aryl is substituted with, for example, one or more halogens or halogen-containing molecules, one or more hydroxyl groups, one or more acyl groups, one or more acyloxy groups, one or more alkoxy groups, one or more aryl groups, one or more carboxy groups, one or more carbonyl groups, one or more alkylcarboxy groups, one or more alkylsulfonoxy groups, one or more alkylcarbonyl groups, one or more nitro groups, one or more cyano groups, one or more acylamide groups, one or more phenyl groups, one or more tolyl groups, one or more chlorophenyl groups, one or more alkoxyphenyl groups, one or more halophenyl groups, one or more benzoxazole groups, one or more thiazoline groups, one or more benzimidazole groups, one or more oxazole groups, one or more thiazole groups, one or more indole groups, etc., or any suitable molecule containing combinations thereof. In some embodiments, the alkyl or substituted alkyl can be C1-C30 alkyl. In some embodiments, the alkyl can be branched or unbranched. In some embodiments, the aryl can be heterocyclic, polycyclic, or monocyclic.

[0054]

Chemical formula

[0055] Exemplary oxathiazine-like compounds include the following:

Chemical formula

[0056] In certain embodiments, 2250 (tetrahydro-1,4,5-oxathiazine-4-dioxide or 1,4,5-oxathiazane-4-dioxide) inhibits GAPDH and is used to treat, prevent, suppress, or alleviate at least one sign or symptom of a disease, disorder, condition, or symptom resulting from or related to glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reaction, immune response, excessive angiogenesis, dysfunction of apoptosis in normal cells, and / or impairment of autophagy, including but not limited to those according to the disclosure herein, which is due to or related to GAPDH activity.

[0057] In certain embodiments, the present disclosure provides hydroxymethylamide isethionate as a chemical compound, in a composition, and for administration according to the methods of the present disclosure.

[0058] In certain embodiments, the present disclosure also includes GAPDH bound to one or more of the compounds of the present disclosure. For example, the present disclosure includes a complex or conjugate of GAPDH and one or more of the compounds of the present disclosure described above.

[0059] As used herein, "complex" refers to one or more of the compounds of the present disclosure complexed with GAPDH, and at least one compound of the present disclosure is bound to or sequestered by GAPDH. As used herein, "conjugate" refers to one or more of the compounds of the present disclosure covalently bound to GAPDH.

[0060] In some embodiments, one or more of the above-described compounds may be covalently bound to one or more of the cysteines of GAPDH. In some embodiments, one or more of the above-described compounds may be covalently bound to the catalytic (active site) cysteine-SH of GAPDH, i.e., Cys-152 of GAPDH.

[0061] In certain embodiments, the disclosure includes compounds that hydrolyze in vitro or in vivo to form hydroxymethyl amide isethionate. In some embodiments, such compounds can include compounds of Formula I where R is a group that leaves in aqueous solution. In certain embodiments, the disclosure includes administering a compound to a subject, where the compound hydrolyzes or metabolizes in vivo to form hydroxymethyl amide isethionate. Examples of such compounds include compounds of Formula I where R is a group that leaves in aqueous solution. In certain embodiments, the disclosure includes a method of inhibiting GAPDH by administering to a subject a compound that hydrolyzes or metabolizes in vivo to form hydroxymethyl amide isethionate. In certain embodiments, the disclosure includes a method of inhibiting NFkB (NFκB) by administering a compound of the disclosure. In certain embodiments, the disclosure includes a method of decreasing the expression of Bcl-2 by administering a compound of the disclosure. In certain embodiments, the disclosure includes a method of increasing the expression of Bax by administering a compound of the disclosure.

[0062] In certain embodiments, the invention also relates to a composition, such as a pharmaceutical composition, containing a compound, complex or conjugate described herein (including its pharmaceutically acceptable solution), and an administrable composition, kit, medical device and pharmaceutical container containing the composition of the disclosure.

[0063] As used herein, the terms "effective amount" or "therapeutically effective amount" mean the amount of a subject compound that elicits a biological or medical response in a tissue, system, animal, or human that is sought by a researcher, veterinarian, physician, or other clinician. In one example, a therapeutically effective amount is from about 0.0001 mg / kg to about 10000 mg / kg, from about 0.001 mg / kg to about 5000 mg / kg, from about 0.01 mg / kg to about 1000 mg / kg, from about 0.05 mg / kg to about 750 mg / kg, from about 0.1 mg / kg to about 600 mg / kg, from about 1 mg / kg to about 500 mg / kg, from about 10 mg / kg to about 400 mg / kg, from about 20 mg / kg to about 300 mg / kg, from about 200 mg / kg to about 500 mg / kg, from about 300 mg / kg to about 400 mg / kg, about 250 mg / kg, 300 mg / kg, 400 mg / kg, 420 mg / kg, 450 mg / kg, about 500 mg / kg, or a dosage or range within any of the disclosed ranges of the subject's body weight.

[0064] As used herein, the terms "administering" or "administer" of a compound mean providing the compound of the present invention to an individual in need of treatment in a form capable of being introduced into the body of the individual, for example, intravenously, subcutaneously, intramuscularly, topically, orally, intraperitoneally, ophthalmically, by intravitreal injection, intrathecally, intranasally, intrapulmonary, transdermally, intravitreally, by inhalation, intratracheally, intravitreally, or in combination thereof. In some embodiments, the compounds of the present invention can be administered in therapeutically useful forms and in therapeutically useful amounts including, but not limited to, oral dosage forms such as tablets, capsules, syrups, suspensions; injection dosage forms such as intravenous (IV), intramuscular (IM) or intraperitoneal (IP), intranasal; enteral or parenteral, transdermal dosage forms including creams, gels, powders or patches; oral dosage forms; inhalation powders, sprays, suspensions, etc.; and rectal suppositories.

[0065] Depending on the particular route of administration desired, various pharmaceutically acceptable carriers known in the art can be used. These include solid or liquid fillers, diluents, hydrotropes, surfactants, and encapsulating substances. Any pharmaceutically active material that does not substantially interfere with the activity of one or more oxathiazine-like compounds may be included.

[0066] As used herein, the term "intravenous administration" includes injection, infusion, and other methods of intravenous administration.

[0067] The term "pharmaceutically acceptable," as used herein, means that a carrier, diluent, or excipient must be compatible with the other ingredients of the formulation and not harmful to its recipient.

[0068] In one aspect, the disclosure includes administering one or more compounds of the disclosure alone or in combination with at least one second active agent. For example, in some aspects, the disclosure includes administering one or more compounds of the disclosure to a subject in need thereof together with an anti-angiogenic agent, an anti-autoimmune agent, and / or an anti-neoplastic agent.

[0069] In one aspect, the disclosure includes administering one or more compounds of the disclosure to a subject in need thereof to inhibit GAPDH activity. In one aspect, the disclosure includes a method of inhibiting GAPDH activity in a subject's cells by about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0070] In one aspect, the disclosure includes reducing or inhibiting the production of adenosine triphosphate (ATP) in a subject in need thereof by administering one or more compounds of the disclosure to inhibit GAPDH activity in the subject.

[0071] In one aspect, the present disclosure includes, but is not limited to, treating, preventing, suppressing, or alleviating at least one sign or symptom of a disease, disorder, or condition caused by or associated with GAPDH activity, such as glycolytic disorders, disorders of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, angiogenesis, autoimmune reactions, immune responses, excessive angiogenesis, dysfunction of apoptosis in normal cells, and / or disorders of autophagy, by inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof.

[0072] In some embodiments, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof to irreversibly inhibit GAPDH. In some embodiments, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof to modulate mitochondrial function and protein production to reduce, inhibit, prevent, and / or eliminate cancer stem cells (CSCs). In some embodiments, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof to increase the production or localization of reactive species, such as reactive oxygen species, in tumors and cancerous cells, thereby reducing the viability of cancer cells without affecting normal cells. In some embodiments, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof to induce the restoration of the normal extracellular matrix of the fibrotic tissue surrounding cancer cells / tumors. In some embodiments, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to reduce, inhibit, prevent, and / or remove cytokines. In some embodiments, the present disclosure includes treating a subject having a therapy / condition that results in an increase in cytokine release or cytokine levels by co-administering one or more compounds of the present disclosure to the subject to prevent, inhibit, or reduce the increase in cytokine release or cytokine levels. In some embodiments, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof to reduce, inhibit, prevent, and / or remove cytokines without interfering with the cytotoxicity to target cancer cells in immunotherapies, including but not limited to T cell-induced therapies such as CAR-T and bispecific therapies.

[0073] In some embodiments, the present disclosure includes selecting a subject having cancer, an autoimmune disease, angiogenesis, or another disease, disorder, condition or symptom disclosed herein that is associated with GAPDH, and administering to the selected subject one or more GAPDH inhibitors comprising an oxathiazine-like compound of the present disclosure. The present disclosure includes methods and compositions for treating a subject having cancer, an autoimmune disease, angiogenesis, or another disease, disorder, condition or symptom disclosed herein.

[0074] In some embodiments, the present disclosure includes methods and compositions for treating cancer, autoimmune disease, neovascularization and / or excessive angiogenesis associated with GAPDH in a subject, comprising administering to the subject one or more GAPDH inhibitors comprising an oxathiazine-like compound of the present disclosure.

[0075] In some embodiments, the present disclosure includes methods for selecting a subject having cancer, autoimmune disease, neovascularization and / or excessive angiogenesis associated with GAPDH for treatment with one or more oxathiazine-like compounds, comprising detecting GAPDH in a biological sample of the subject and selecting the subject for treatment with one or more oxathiazine-like compounds of the present disclosure. In some embodiments, cancer, autoimmune disease, neovascularization and / or excessive angiogenesis associated with GAPDH in a subject is determined by isolating a sample of cells or a biological sample from the subject and assessing GAPDH activity in the cell or biological sample.

[0076] In some embodiments, the present disclosure includes methods for screening for GAPDH inhibition by one or more oxathiazine-like compounds, comprising contacting a cell or biological sample containing GAPDH with one or more oxathiazine-like compounds, determining whether GAPDH is inhibited in the cell or biological sample, and selecting at least one compound that inhibits GAPDH from the one or more oxathiazine-like compounds. In some embodiments, GAPDH inhibition that exceeds a threshold value (e.g., at least 30% above a control) indicates that the compound has anti-cancer, anti-autoimmune, anti-neovascularization and / or anti-excessive angiogenesis activity.

[0077] In some embodiments, the disclosure includes a method of determining whether GAPDH is inhibited by one or more oxathiazine-like compounds by contacting a cell or biological sample containing GAPDH with the one or more oxathiazine-like compounds and determining whether GAPDH is inhibited in the cell or biological sample.

[0078] In some embodiments, the disclosure is a method of evaluating the anti-cancer, autoimmunity, angiogenesis and / or hyper-angiogenesis properties of an oxathiazine-like compound for the treatment of cancer, autoimmune diseases, angiogenesis and / or hyper-angiogenesis, the method comprising contacting a cell or biological sample with the oxathiazine-like compound and determining whether GAPDH is inhibited in the cell or biological sample, wherein inhibition of GAPDH by the oxathiazine-like compound indicates that the oxathiazine-like compound is useful for the treatment of cancer, autoimmune diseases, angiogenesis and / or hyper-angiogenesis.

[0079] The anti-GAPDH agents of the present disclosure can be administered to a subject at risk of or suffering from various diseases, disorders, and conditions. Such diseases, disorders, and conditions may be characterized by angiogenesis and / or excessive angiogenesis. The present disclosure also provides methods and compositions for modulating and regulating angiogenesis, modulating and regulating angiogenesis, and preventing, treating, suppressing, or alleviating angiogenesis and / or excessive angiogenesis, also referred to as angiogenesis-related or neovascularization-related diseases, disorders, and conditions. Non-limiting examples of such diseases, disorders, and conditions include tumors, cancers, skin diseases (including psoriasis, telangiectasia, wound granulation, scleroderma, angiogenesis as a result of infections (such as cat scratch disease, bacterial ulcers, etc.), but not limited thereto), macular degeneration or age-related blindness, diabetic ulcers, chronic ulcers and wounds, stroke, traumatic brain injury, retinal neovascularization, corneal neovascularization (such as those caused by trachoma, infections, inflammation, transplantation, or trauma, etc.), diabetic retinopathy, diabetic retinal edema, diabetic macular edema, ischemic retinopathy, hypertensive retinopathy, obstructive retinopathy, retinopathy of prematurity, neovascularization after trauma, neovascularization after infection, neovascularization after transplantation, neovascularization after retinal detachment or retinal degeneration, neovascular glaucoma, anterior chamber and / or anterior chamber angle neovascularization, choroidal neovascularization (CNV), subretinal neovascularization, retrolental fibroplasia, ocular histoplasmosis syndrome, myopic degeneration, retinitis pigmentosa, uveitis, rubeosis, retrolental fibroplasia, ocular histoplasmosis, and idiopathic central serous chorioretinopathy, amyotrophic lateral sclerosis, sarcoidosis, scleroderma, lupus, Parkinson's disease, sclerosis, Stevens-Johnson syndrome, neoplasms, von Willebrand disease, vasculitis, and one or more of Kawasaki disease.

[0080] The present disclosure also provides methods and compositions for treating subjects suffering from cardiovascular diseases including, but not limited to, atherosclerosis, restenosis, atherosclerotic plaques, and hemangiomas. Atherosclerosis is a form of chronic vascular injury in which some of the properties of normal vascular smooth muscle cells (VSMCs) in the arterial wall change and a dense capillary network develops in atherosclerotic plaques. These fragile microvessels can bleed, leading to blood clotting and subsequent reduction in blood flow to the myocardium and risk of heart attack. Restenosis typically occurs after coronary artery bypass surgery, endarterectomy, and heart transplantation, particularly after balloon angioplasty, atherectomy, laser ablation, or intravascular stent placement. This is accompanied by extensive growth of microvessels. The methods provided herein are useful for treating these cardiovascular diseases by inhibiting angiogenesis in cardiovascular tissue.

[0081] In one aspect, the present disclosure relates to the treatment of macular degeneration. In particular, an ophthalmic formulation containing a compound of the present disclosure is administered to a subject in need thereof. Ophthalmic indications according to the present disclosure include all forms of diabetic retinopathy, particularly diabetic macular edema, in people with or without diabetic macular edema. Diabetic retinopathy is a serious condition that affects millions of people. In one aspect, the composition of the present disclosure is administered by intravitreal injection.

[0082] In some aspects, the present disclosure includes inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need of reducing, suppressing, and / or preventing angiogenesis and / or excessive angiogenesis in the subject. In some aspects, at least one sign or symptom can include rash, muscle pain, joint pain, fatigue, anemia, inflammation, abdominal pain, abdominal distension, diarrhea, nausea, acid reflux, weight gain, fever, persistent headache, hemorrhagic complications (e.g., bleeding), hypertension, hypotension, leukopenia, tumor growth, cachexia, photosensitivity, eye redness, eye irritation, or combinations thereof.

[0083] In one aspect, the present disclosure includes preventing, suppressing, or reducing at least one side effect of a drug administered to a subject suffering from a disease, disorder, or condition caused by or associated with angiogenesis and / or excessive angiogenesis by inhibiting GAPDH activity by co-administering one or more oxathiazine-like compounds to the subject. In some aspects, the at least one side effect may include one or more of hemorrhagic complications (e.g., bleeding), hypertension, diarrhea, malaise, cytopenia, impaired wound healing, skin itching, dryness or peeling, dry eye or watering eyes, pain, headache, rash, dizziness, weight loss, hair loss, swelling, abnormal bruising, seizures, muscle weakness, numbness, infections, fever, chills, aches, pains, loss of appetite, changes in weight, joint pain / swelling, or combinations thereof.

[0084] In one aspect, the present disclosure includes methods and compositions for increasing the therapeutic index of a chemotherapeutic agent (e.g., reducing toxicity, increasing tumor uptake of the drug, increasing efficacy) by inhibiting GAPDH activity by co-administering one or more oxathiazine-like compounds of the present disclosure with the chemotherapeutic agent. In some aspects, the chemotherapeutic agent may include trastuzumab, alemtuzumab, bevacizumab, blinatumomab, brentuximab vedotin, infliximab, eculizumab, certolizumab, daclizumab, cetuximab, denosumab, dinutuximab, ibritumomab tiuxetan, ipilimumab, nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, trastuzumab. In some aspects, this combination increases the therapeutic index by lowering the toxicity of the combination therapy. The lower the toxicity, the more chemotherapeutic agent(s) can be delivered while maintaining acceptable side effects. It is also contemplated that because the combination therapy is more effective, fewer chemotherapeutic agents can be used to obtain the same results as were provided by previous compositions.

[0085] As used herein, the phrases "administered concomitantly" or "administered in combination" mean that two or more agents are administered concurrently. Concomitant administration or combination can be achieved by mixing the two agents in a single formulation or by administering the two agents separately but simultaneously or at short intervals. For example, generally, the two agents are administered concomitantly within a time range of 6 hours to 168 hours. In this case, the agents can be administered in either order, i.e., the chemotherapeutic agent can be administered first, or one or more of the oxathiazine-like compounds of the present disclosure can be administered first. In some embodiments, the two agents are administered concomitantly in a single formulation or sequentially and separately.

[0086] In one aspect, the present disclosure relates to a method of reducing chemotherapy-related toxicity in a patient being treated with a chemotherapeutic agent and at risk of such toxicity, the method comprising treating the patient with one or more oxathiazine-like compounds and a chemotherapeutic agent such that the risk of chemotherapy-related toxicity in the patient is reduced. In one embodiment, the chemotherapy-related toxicity is cardiotoxicity, nephrotoxicity, hepatotoxicity, pulmonary toxicity, cutaneous toxicity or gastrointestinal toxicity. For example, some chemotherapeutic agents containing anthracyclines can cause direct damage to the heart (either acute or chronic). Chemotherapeutic agents containing cisplatin, cyclophosphamide and ifosfamide cause uro / renal toxicity. Drugs with pulmonary toxicity containing bleomycin can have a profound impact on the lungs. Cutaneous toxicity is also common with chemotherapeutic agents and includes transient rash (carmustine, cytarabine, gemcitabine, asparaginase and procarbazine), photosensitivity (mitomycin, 5-FU, methotrexate, vinblastine and dacarbazine), dermatitis, hyperpigmentation, urticaria, nail changes, alopecia and radiation recall. Gastrointestinal toxicity, including stomatitis or diarrhea, is also common.

[0087] In some embodiments, the patient has cancer or a tumor, including but not limited to: biliary tract cancer; brain cancer including glioblastoma and medulloblastoma; breast cancer; triple negative breast cancer; uterine cancer; fallopian tube cancer; cervical cancer; choriocarcinoma; colon cancer; bladder cancer; endometrial cancer; retinoblastoma; vaginal cancer; vulvar cancer; esophageal cancer; oral cancer (mouth cancer); gastric cancer; kidney cancer; hematological tumors including acute lymphocytic and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer (hepatic carcinoma); lung cancer; head and neck cancer or oral cancer (oral cavity, pharynx, esophagus, nasopharynx, jaw, tonsil, nose, lip, salivary gland, tongue, etc.); lymphomas including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; neuroendocrine tumors; oral cancer including squamous cell carcinoma; adrenal cancer; anal cancer; angiosarcoma; appendiceal cancer; cholangiocarcinoma; bone cancer; carcinoid tumors; soft tissue sarcomas; rhabdomyosarcoma; eye cancer; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells as well as fallopian tube cancer; gallbladder cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer including germ cell tumors (seminoma, non-seminoma (teratoma, choriocarcinoma)), stromal tumors, and embryonal cell tumors; penile cancer; angioendothelioma; gastrointestinal cancer; ureteral cancer; urethral cancer; spinal cord cancer; pituitary cancer; primary central nervous system (CNS) lymphoma; thyroid cancer including goiter and medullary carcinoma; and kidney cancer including adenocarcinoma and Wilms tumor. In some embodiments, the cancer or tumor includes breast cancer, prostate cancer, colorectal cancer, lymphoma, multiple myeloma, and melanoma.

[0088] The toxicity and therapeutic efficacy of such molecules can be determined by standard pharmacological procedures in cell culture or experimental animals, such as determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose having a therapeutic effect on 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio of LD 50 / ED 50 and can be expressed as the ratio of LD / ED.

[0089] As used herein, the term "therapeutic index" with respect to a chemotherapeutic agent indicates the safety of the chemotherapeutic agent. In some embodiments, the therapeutic index may include a comparison of the amount of therapeutic agent that produces a therapeutic effect (e.g., killing of cancer cells) and the amount of therapeutic agent that causes toxicity (e.g., liver toxicity). According to certain embodiments, but not limited to, (1) when increasing the dosage of a chemotherapeutic agent above the current therapeutic dosage, (2) when keeping the dosage of a chemotherapeutic agent the same as the current therapeutic dosage, or (3) when decreasing the dosage of a chemotherapeutic agent below the current therapeutic dosage, it is contemplated that an improvement in the therapeutic index may occur using the compositions and / or methods described herein. In some embodiments, the compositions and methods that include the scenarios of this paragraph can elicit an improved or the same therapeutic effect as seen at the current therapeutic dosage with less or no worsening of toxicity.

[0090] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering to a subject in need thereof one or more compounds of the present disclosure, thereby preventing angiogenesis in the subject by downregulating angiogenesis by preventing unwanted excessive angiogenesis in the subject.

[0091] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering to a subject in need thereof one or more compounds of the present disclosure, thereby preventing unwanted excessive angiogenesis in the subject by downregulating angiogenesis by preventing unwanted excessive angiogenesis in the subject.

[0092] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering to a subject in need thereof one or more compounds of the present disclosure, thereby suppressing glycolysis disorder by preventing unwanted excessive angiogenesis in the subject by preventing unwanted excessive angiogenesis in the subject.

[0093] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, thereby preventing unwanted excessive angiogenesis in the subject, and thereby preventing, suppressing, reducing or reversing a disorder of the proteolytic pathway.

[0094] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, thereby preventing unwanted excessive angiogenesis in the subject, and thereby suppressing uncontrolled protein aggregation.

[0095] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, thereby preventing unwanted excessive angiogenesis in the subject, and thereby suppressing aerobic glycolysis.

[0096] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, thereby suppressing mitochondrial dysfunction.

[0097] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, thereby preventing unwanted excessive angiogenesis in the subject, and thereby suppressing an increase in glucose uptake or metabolism.

[0098] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, by administering one or more oxathiazine-like compounds to the subject, thereby preventing unwanted excessive angiogenesis in the subject and suppressing an autoimmune response thereby.

[0099] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, by administering one or more oxathiazine-like compounds to the subject, thereby preventing unwanted excessive angiogenesis in the subject and suppressing an immune response thereby.

[0100] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, by administering one or more oxathiazine-like compounds to the subject, thereby preventing unwanted excessive angiogenesis in the subject and suppressing dysfunction of apoptosis of normal cells thereby.

[0101] In one aspect, the present disclosure includes a method of inhibiting GAPDH activity by administering one or more compounds of the present disclosure to a subject in need thereof, by administering one or more oxathiazine-like compounds to the subject, thereby preventing unwanted excessive angiogenesis in the subject and suppressing impairment of autophagy thereby.

[0102] In one aspect, the present disclosure includes inhibiting, reducing, or preventing GAPDH activity by administering one or more oxathiazine-like compounds, wherein the one or more oxathiazine-like compounds interact with the reactive (catalytic) cysteine-SH of the active center of GAPDH of the subject, thereby inactivating GAPDH of the subject.

[0103] In some aspects, the present disclosure includes reducing the catalytic activity of GAPDH in a subject in a dose- and time-dependent manner. For example, inhibition of GAPDH by the compounds of the present disclosure may be due to inactivation of the enzyme, e.g., by covalent interaction with the catalytic cysteine of GAPDH. This interaction has a great impact on the pharmacokinetics and dosing schedule of the compounds of the present disclosure in a patient. In some aspects, once covalently inactivated, GAPDH activity can only be restored by synthesis of new enzyme protein. Thus, the duration of target inhibition is determined by the half-life of the GAPDH enzyme. Measurement of the blood levels of the free compounds of the present disclosure that are metabolized and excreted is outdated as an indicator of target inhibition. In some aspects, the blood levels of the compounds of the present disclosure administered to a patient do not reflect the active state of the enzyme due to this phenomenon. The duration of enzyme inhibition far exceeds the presence of the free compounds of the present disclosure in the blood. For this reason, the dosing interval of the compounds of the present disclosure is based on the half-life of the GAPDH enzyme protein.

[0104] In one aspect, a patient is treated with one or more oxathiazine-like compounds or combinations thereof administered intravenously, orally, or a combination thereof. In one aspect, a patient is treated with 2250 (also referred to as "Compound 2250", "C-2250", or "GP-2250") administered intravenously, orally, or a combination thereof.

[0105] In one aspect, one or more therapeutic agents for treating a subject having a disease, disorder or condition caused by or related to glycolytic disorders, disorders of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reactions, immune responses, excessive angiogenesis, dysfunction of apoptosis in normal cells, and / or disorders of autophagy, such as anti-VEGF antibodies, bevacizumab, ranibizumab, brolucizumab, lapatinib, sunitinib, sorafenib, axitinib, cabozantinib, lenvatinib, ponatinib, ramucirumab, regorafenib, vandetanib, pazopanib, pegaptanib, bevaciranib, aflibercept, thiazolidinedione, conbercept and ranparizumab, corticosteroids, immunosuppressive agents such as cyclosporine, tacrolimus, anti-inflammatory agents such as dimethyl fumarate, sphingosine-1-phosphate (S1P) receptor modulators such as siponimod, fingolimod, selumetinib, ozanimod, ponesimod, autoimmune modulator peptides such as glatiramer acetate and similar random-sized peptides, biological drugs such as antibodies, fusion proteins and interferon-based drugs, are administered to a patient in combination with one or more oxathiazine-like compounds or combinations thereof.

[0106] In some aspects, the disclosure includes administering one or more oxathiazine-like compounds in combination with one or more of tocilizumab, antihistamines, antipyretics, anti-inflammatory compounds, corticosteroids, glucocorticoids, TNF inhibitors (such as etanercept), siltuximab, T cell depletion antibody therapies such as alemtuzumab and anti-thymocyte globulin (ATG), IL-1R family inhibitors (anakinra), ibrutinib, and cyclophosphamide.

[0107] The compounds according to the present invention can be administered by any suitable method. Solid dosage forms for oral administration include capsules, tablets, pills, powders, orally disintegrating tablets, and granules. In such solid dosage forms, the composition to be administered is mixed with at least one inert pharmaceutically acceptable excipient and / or filler or bulking agent (such as starch, lactose, sucrose, glucose, mannitol, and silicic acid), binder (such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and gum arabic), humectant (such as glycerol), disintegrant (such as agar, calcium carbonate, potato starch, tapioca starch, alginic acid, certain silicates, and sodium carbonate), dissolution retardant (such as paraffin), absorption promoter (such as quaternary ammonium compounds), wetting agent (such as cetyl alcohol and glycerol monostearate), absorbent (such as kaolin and bentonite clay), and lubricant (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain a buffering agent.

[0108] Solid compositions of the same type can be employed as fillers in soft and / or hard gelatin capsules using excipients such as lactose or milk sugar, and fillers such as high molecular weight polyethylene glycol. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other coatings known in the pharmaceutical formulation art. These may optionally contain opacifying agents and may be compositions that release the given composition(s) optionally with delay, only in or targeted to a particular part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of the same type can be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugar, and fillers such as high molecular weight polyethylene glycol.

[0109] In certain embodiments, the capsule may contain an excipient formulation containing one or more of hydroxypropyl methylcellulose (HPMC), gelatin, meglumine, and fish gelatin. In certain embodiments, the capsule may contain compound 2250 in combination with tauroursodeoxycholic acid and / or taurine. The capsule may optionally further contain one or more of lycopene, ellagic acid (polyphenol), curcumin, piperine, delphinidin, resveratrol, isothiocyanates such as sulforaphane, capsaicin, and piperlongumine.

[0110] When the compound of the invention according to the claims is used in the form of microparticles or nanoparticles, higher blood levels can be achieved. The present invention includes microparticles and / or nanoparticles of the compounds of the present disclosure in tablet form or encapsulated in capsules.

[0111] In certain embodiments, the present disclosure relates to orally administering an oxathiazine-like compound to a patient. In some embodiments, the oxathiazine-like compound is formulated into a capsule or tablet. In certain embodiments, the oral dosage form contains about 50 mg to 1000 mg of the oxathiazine-like compound. In certain embodiments, the oral dosage form contains about 100 mg to 500 mg of the oxathiazine-like compound. In certain embodiments, the oral dosage form contains about 200 mg to 400 mg of the oxathiazine-like compound. In certain embodiments, the oral dosage form contains about 250 mg to 350 mg of the oxathiazine-like compound. In certain embodiments, the oxathiazine-like compound is C-2250.

[0112] In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 0.01 μg / ml to about 500 μg / ml. In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 0.1 μg / ml to about 100 μg / ml. In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 10 μg / ml to about 50 μg / ml.

[0113] In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 0.001 wt% to about 5 wt%, about 0.01 wt% to about 3.5 wt%, about 0.1 wt% to about 3 wt%, about 0.5 wt% to about 2.5 wt% or about 1 wt% to about 2 wt%. In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 0.01% to about 1.5%. In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 0.1% to about 1%. In some embodiments, the oxathiazine-like compound is provided in the composition at a concentration of about 100 μM to about 5000 μM, about 250 μM to about 2500 μM, about 500 μM to about 2000 μM, about 750 μM to about 1500 μM, about 1000 μM to about 1250 μM, or any other concentration within the recited ranges.

[0114] In some embodiments, the oxathiazine-like compound is provided in a unit dosage form of the composition. As used herein, "unit dosage form" is a composition containing an amount of the oxathiazine-like compound suitable for administration in a single dose to an animal such as a mammal, e.g., a human subject, in accordance with proper medical practice. These compositions can contain from about 0.1 mg (milligram) to about 500 mg, such as from about 5 mg to about 350 mg of the oxathiazine-like compound. The frequency of treatment with the compositions of the present invention can be varied so that the desired target plasma level is achieved and maintained. For this purpose, non-limiting examples of treatment schedules include daily, twice a day, three times a day, weekly, bi-weekly, monthly, and combinations thereof. Alternatively, the compositions of the present invention can also be administered as one or more different continuous infusions, for example, following a bolus, where the rate and amount of the drug administered are different, and such regimens are optionally interrupted by one or more additional bolus injections.

[0115] In certain embodiments, one or more oxathiazine-like compounds of the present disclosure are administered to a subject prior to administration of a therapeutic agent that is expected to lead to glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, angiogenesis, autoimmune reaction, immune response, excessive angiogenesis, impairment of the apoptotic function of normal cells, and / or impairment of autophagy in the subject. For example, in one embodiment, one or more oxathiazine-like compounds of the present disclosure are administered to the subject about 12 hours to 96 hours, such as 24 hours, 48 hours, or 72 hours, prior to administration of a therapeutic agent that is expected to lead to (e.g., directly or indirectly cause or promote) glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, angiogenesis, autoimmune reaction, immune response, excessive angiogenesis, impairment of the apoptotic function of normal cells, and / or impairment of autophagy in the subject. In one embodiment, one or more oxathiazine-like compounds of the present disclosure are administered once or multiple times prior to administration of a therapeutic agent that is expected to lead to glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, angiogenesis, autoimmune reaction, immune response, excessive angiogenesis, impairment of the apoptotic function of normal cells, and / or impairment of autophagy in the subject. In certain embodiments, one or more oxathiazine-like compounds of the present disclosure are administered to the subject simultaneously with a therapeutic agent that is expected to lead to glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, angiogenesis, autoimmune reaction, immune response, excessive angiogenesis, impairment of the apoptotic function of normal cells, and / or impairment of autophagy in the subject.In certain embodiments, an oxathiazine-like compound is administered to a subject within about 1 hour to about 24 hours, about 4 hours to about 18 hours, about 6 hours to about 15 hours, or about 8 hours to about 12 hours after administration of a therapeutic agent that is expected to promote glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reaction, immune reaction, excessive angiogenesis, impairment of the apoptosis function of normal cells, and / or impairment of autophagy in the subject.

[0116] In certain embodiments, one or more oxathiazine-like compounds of the present disclosure are administered according to a regimen during a period in which glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reaction, immune reaction, excessive angiogenesis, impairment of the apoptosis function of normal cells, and / or impairment of autophagy angiogenesis is expected to occur. For example, in one embodiment, one or more oxathiazine-like compounds of the present disclosure are administered daily, every other day, every other week, or weekly to a patient throughout their lifetime, until remission, for a period of several years, several months, 2 weeks to 12 weeks, 3 weeks to 10 weeks, or 4 weeks to 8 weeks, before, during, and / or after administration of a therapeutic agent that is expected to lead to glycolytic impairment, impairment of the proteolytic pathway, uncontrolled protein aggregation, aerobic glycolysis, mitochondrial dysfunction, increased glucose uptake or metabolism, neovascularization, autoimmune reaction, immune reaction, excessive angiogenesis, impairment of the apoptosis function of normal cells, and / or impairment of autophagy in the subject.

[0117] In one aspect, one or more oxathiazine-like compounds are provided in a composition and administered to a subject in need thereof at a total daily dose of about 0.001 g to about 1000 g, such as about 0.01 g to about 500 g, 0.1 g to 300 g, 0.5 g to 200 g, 1 g to 100 g, or any amount within the recited ranges. The daily dose can be administered in the form of an orally administrable composition. The daily dose may be administered in the form of capsules, tablets or a pharmaceutically acceptable solution. The daily dose may be administered in a form containing compound 2250 at a concentration of about 0.01% to about 5% (w / v), about 0.1% to about 3% (w / v), about 0.5% to about 2.5% (w / v) or about 1% to about 2% (w / v).

[0118] The daily dose may be administered in a form containing one or more oxathiazine-like compounds at a concentration of about 0.001 μg / ml to about 1000 μg / ml, about 0.01 μg / ml to about 750 μg / ml, about 0.05 μg / ml to about 500 μg / ml, about 0.1 μg / ml to about 300 μg / ml, about 0.5 μg / ml to about 200 μg / ml, about 1 μg / ml to about 100 μg / ml, about 5 μg / ml to about 50 μg / ml, about 10 μg / ml to about 25 μg / ml or about 15 μg / ml to about 20 μg / ml. The daily dose may be administered in a form containing one or more solubilizing agents, such as polyols.

[0119] The effective dose of the oxathiazine-like compound is provided in a composition and may include dosage units containing about 0.01 mg / kg to 500 mg / kg, about 1 mg / kg to 100 mg / kg, or about 5 mg / kg to 50 mg / kg of the oxathiazine-like compound per day. In some aspects, the dosage units are administered every other day, every other week or weekly.

[0120] The specific effective dosage for any particular patient will be determined by a variety of factors including neovascularization and / or angiogenesis, the severity or likelihood of the disorder or disease; the activity of the particular compound used; the age, weight, general health, sex and diet of the patient; the preparation of the particular compound; the time and route of administration; the duration of administration; therapeutic agents used in combination with or simultaneously with the particular compound employed; and like factors known in the medical arts. The effective dosage may be changed over time in response to the amelioration or worsening of a GAPDH-mediated disorder, disease or condition. For chronic conditions, an effective dosage can be administered to the subject for several days, weeks, months, years or over the lifetime of the subject. The number and frequency of administrations or co-administrations can vary depending on the likelihood or severity of the GAPDH-mediated disorder, disease or condition, and the specific response of the patient to the particular compound and / or second therapeutic agent being administered to the subject.

[0121] In another aspect, the disclosure provides a method, kit, apparatus or device for a screening assay to identify additional inhibitors of GAPDH. One or more test compounds can be assayed for binding to GAPDH and inhibition of GAPDH. In one aspect, the disclosure includes combining a test compound with a suitable buffer or solvent, e.g., a buffer or solvent that dissolves the test compound, contacting the test compound with recombinant GAPDH in the buffer to form a reaction mixture, and subjecting an aliquot of the reaction mixture to an enzyme activity assay to identify a compound that inhibits GAPDH.

[0122] In some embodiments, the enzyme activity assay can be performed using a recombinant GAPDH probe that detects changes in NAD+ concentration in a multi-well plate compared to a control solvent. In some embodiments, the enzyme activity assay can include a sodium pyrophosphate buffer. In some embodiments, the recombinant GAPDH probe may be incubated with sodium arsenite, NAD+, and glyceraldehyde-3-phosphate (G3P). Enzyme activity can be measured, for example, at room temperature using a microplate reader spectrophotometer as an increase in absorbance at 340 nm due to the reduction of NAD+. In some embodiments, the recombinant GAPDH may first be diluted with a sodium pyrophosphate buffer, for example, up to a volume of 100 μl. Subsequently, an additional 100 μl reaction mix containing sodium arsenite, NAD+, and G3P can be quickly added to each well using a repeat pipettor, the plate may be mixed in the plate reader, for example, for 5 seconds, and then absorbance measurements are taken. In some embodiments, absorbance may be measured once every 10 to 20 seconds over a period of 20 minutes, and the rate is calculated from the change in absorbance in the linear phase. Inhibition of GAPDH is indicated by a decrease in the rate of NAD+ reduction compared to the control solvent.

[0123] In one embodiment, the present disclosure provides a method for identifying a test compound that inhibits GAPDH, comprising combining a test compound and a solvent to form a solution, contacting the solution with recombinant GAPDH in a buffer to form a reaction mixture, subjecting an aliquot of the reaction mixture to an enzyme activity assay, detecting a change in NAD+ concentration in the enzyme activity assay, and identifying a test compound that reduces the NAD+ concentration in the enzyme activity assay compared to a control solvent.

[0124] In another aspect, the present disclosure provides a method, kit, apparatus or device for providing biomarkers for clinical use. In some aspects, the present disclosure provides biomarkers for use in patients having or at risk of having cancer. In some aspects, the present disclosure provides a method of using GAPDH as a biomarker by obtaining peripheral blood mononuclear cells (PBMCs) from a subject, lysing the PBMCs, and monitoring GAPDH activity in the lysed PBMCs. In some aspects, the method comprises subjecting the PBMC lysate to an enzyme activity assay, detecting a change in NAD+ concentration in the enzyme activity assay, and monitoring inhibition of GAPDH by an administered GAPDH inhibitor based on a decrease in NAD+ concentration in the enzyme activity assay compared to a control solvent.

[0125] Inhibition of GAPDH in peripheral blood mononuclear cells (PBMCs) can function as a biomarker for the state of GAPDH inhibition in cancerous tissue. Similar to cancer tissue, the compounds of the present disclosure can covalently inhibit GAPDH in PBMCs. However, in contrast to cancer cells, GAPDH is not rate-limiting in PBMCs and is not harmful to these cells. The half-life of the GAPDH protein in PBMCs is estimated to be the same as or similar to the half-life in the patient's cancer tissue. The degree of inhibition of GAPDH in PBMCs can directly reflect the active state of GAPDH in the target tissue.

[0126] In some embodiments, the present disclosure obtains peripheral blood mononuclear cells (PBMCs) from a subject, lyses the PBMCs, monitors the GAPDH activity in the lysed PBMCs, subjects the lysed PBMCs to an enzyme activity assay, detects a change in the NAD+ concentration in the enzyme activity assay, monitors the inhibition of GAPDH by the administered GAPDH inhibitor based on a decrease in the NAD+ concentration in the enzyme activity assay compared to a control solvent, determines the degree of inhibition of GAPDH in the PBMCs, and when the degree of inhibition of GAPDH by a particular compound exceeds a predetermined threshold, such as about 50%, about 60%, about 70%, about 80%, about 90% or about 95%, identifies a subject as a candidate suitable for treatment with a particular GAPDH inhibitor compound of the present disclosure, thereby providing a method for tracking the degree of GAPDH inhibition in a patient treated with one or more compounds of the present disclosure.

Example

[0127] The embodiments of the present disclosure will be further described with reference to the following examples, which are presented for illustrative purposes only and should not be used to limit the scope of the invention or interpret the invention.

[0128] Example 1 Various oxathiazine-like compounds of the present disclosure are synthesized and assayed for their interaction with GAPDH. Isethionic acid amide and methylene glycol were identified as hydrolysis products. The reactive transient reaction intermediate is hydroxymethyl isethionate amide. This intermediate interacts covalently with the reactive cysteine-SH in the active center of GAPDH, inactivating the enzyme. The covalently labeled enzyme is purified and the reaction intermediate is identified and elucidated using various analytical methods including mass spectrometry of the labeled peptide.

[0129] Example 2 The inhibition of LPS-stimulated cytokine release by the compounds of the present disclosure is assayed and found to be higher under high glucose (10 mM) compared to low glucose (0.5 mM). Hepte lidic acid is the positive control.

[0130] Example 3 When lactate production is used as a surrogate measure, the effect of the compounds of the present disclosure on LPS stimulation is accompanied by a decrease in lactate. LPS stimulation results in an increase in Warburg-like glycolysis. GAPDH becomes rate-limiting only under such high glycolysis conditions.

[0131] Example 4 Recombinant GAPDH is directly inhibited by 2250. Since it is a cell-free assay, incubation time may be important. The specific doses required for half-maximal or complete inhibition of GAPDH are tested in vitro and in vivo in rodents. These in vitro and in vivo data provide a measure related to the target doses required to inhibit GAPDH to varying degrees in tissues, such as cancer tissues, which is a more direct measure of the effect of the compounds of the present disclosure, as opposed to cell assays such as induction of apoptosis or ROS production.

[0132] The extent of GAPDH occupancy by the compounds of the present disclosure in patients is directly detected using a PET-compatible derivative of the compounds of the present disclosure, for example, by incorporating Fluor 18.

[0133] Example 5 GAPDH is the rate-limiting glycolytic enzyme in cells that operate under aerobic glycolysis conditions such as tumor cells. Therefore, partial inhibition is expected to disrupt the energy metabolism of tumor cells. This is in contrast to normal cells, whose energy metabolism is mainly based on oxidative phosphorylation. GAPDH is not the rate-limiting enzyme of glycolysis in normal cells, and partial inhibition of GAPDH is tolerated. The degree of GAPDH enzyme activity was tested against a control using 2250 at concentrations of 100 μM and 250 μM. The results are shown in Figure 1, which shows inhibition of GAPDH enzyme activity. The effect of treatment with GP-2250 (100 μM and 250 μM) on the activity of recombinant GAPDH (rGAPDH) was tested using the Glyceraldehyde-3-phosphate Dehydrogenase Activity Assay Kit (ab204732 from Abcam) after incubation at 37 °C for up to 60 minutes. rGAPDH activity was inhibited up to 40% maximally, dose- and time-dependently, by 100 μM and 250 μM of GP-2250, compared to the untreated control. The control value at the 60-minute time point decreased slightly compared to the 30-minute time point due to the thermal instability of the enzyme. The curves of GP-2250 are measurement data not normalized against the control. Data are presented as mean ± S.D.

[0134] The GAPDH activity assay using recombinant protein shows a significant inhibition of activity, dose- and time-dependently. It is noteworthy that partial inhibition by GP-2250 is sufficient to disrupt the energy metabolism of tumor cells. This is demonstrated by the decrease in ATP achieved by the concentration of GP-2250 corresponding to the concentration required for partial GAPDH inhibition (see Figure 4).

[0135] Example 6 The effects of treatment with various concentrations of GP-2250 on ROS formation were tested in two pancreatic cancer cell lines, a) PancTuI and b) BxPC3, as shown in Figure 2. In both cell lines, the increase in ROS was concentration-dependent. The BxPC3 cell line was more sensitive to ROS formation than PancTuI. The somewhat higher concentration of GP-2250 (500 μM or more) required for ROS formation is most likely due to the short incubation time of 90 minutes in this cell assay. A higher potency of GP-2250 is expected with a longer incubation time.

[0136] Figure 2 shows ROS formation. The effect of treatment with the indicated concentration of GP-2250 on ROS formation was tested in two pancreatic cancer cell lines, a) PancTuI and b) BxPC3, using a fluorescence ROS detection assay (ROS / Superoxide Detection Assay Kit, Abcam (ab139476)) after incubation with GP-2250 for 90 minutes at 37°C. The negative control (NC+NAC) contained N-acetylcysteine (NAC; 5 mM) in addition to the ROS inhibitor that is part of the Assay Kit. Untreated control (U). Data are presented as mean ± S.D. Significance levels calculated compared to the untreated control (U). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0137] Example 7 The level of ATP in tumor cells is regarded as a measure of the effect of GP-2250 on its energy metabolism. ATP was tested in the PancTuI cell line at 3 hours, 6 hours, and 24 hours as shown in Figure 3. ATP was also tested in the BxPC3 cell line at 3 hours, 6 hours, and 24 hours as shown in Figure 4. In both cell lines analyzed, the amount of ATP decreased in response to the concentration of GP-2250 and the incubation time. The decrease in ATP was already apparent at 250 μM at 3 hours in PancTuI and at 6 hours in the less sensitive BxPC3. Since partial inhibition of GAPD is caused at this concentration (Figure 1), GAPD inhibition is associated with the decrease in ATP, which itself becomes a signal sufficient to induce apoptosis. The decrease in ATP is not due to cytotoxicity. Any impairment of cell viability by GP-2250 required a concentration higher than that required to decrease ATP.

[0138] Figure 3 shows the decrease in ATP in the PancTuI cell line. Effect of treatment with the indicated concentration of GP-2250 on the amount of ATP (thick bars) compared to cell viability (thin bars) after incubation at 37 °C for a) 3 hours, b) 6 hours, and c) 24 hours. The strong decrease in ATP reflects the impairment of energy metabolism by GP-2250. Since the decrease in ATP precedes the decrease in cell viability, it is not due to an impairment of cell viability. ATP was measured using a luminescence detection kit (ab113849 from Abcam), and cell viability was measured using an MTT assay (M5655 from Sigma). Data are shown as changes (%) relative to untreated control (NC) and presented as mean ± S.D. Significance levels compared to NC. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0139] Figure 4 shows the decrease in ATP in the BxPC3 cell line. The effect of treatment with the indicated concentrations of GP-2250 on the amount of ATP (thick bars) compared to cell viability (thin bars) after incubation at 37°C for a) 3 hours, b) 6 hours, and c) 24 hours. The strong decrease in ATP reflects the impairment of energy metabolism by GP-2250. Since the decrease in ATP precedes the decrease in cell viability, it is not due to the impairment of cell viability. ATP was measured using a luminescence detection kit (ab113849 from Abcam), and cell viability was measured using an MTT assay (M5655 from Sigma). Data are shown as changes (%) relative to untreated controls (NC) and presented as mean ± S.D. Significance levels compared to NC. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0140] Example 8 The apoptotic pathway is induced by a decrease in ATP or an increase in ROS. These cause a shift in the balance between the apoptosis-promoting (death) protein Bax and the anti-apoptotic (survival) protein Bcl-2. The mitochondria become destabilized, and the apoptotic caspase cascade seals the apoptotic cell suicide. GP-2250 (200 μM) increased the expression of Bax and decreased the expression of Bcl-2 with increasing incubation time, as shown by Western blot (Figure 5). The increase in Bax and the decrease in Bcl-2 indicate that GP-2250 induces apoptosis via the intrinsic mitochondrial pathway. In addition, the concentration of GP-2250 (200 μM) sufficient to change the Bax / Bcl-2 ratio corresponds to the concentration (250 μM) for partial GAPDH inhibition (Figure 1). This finding associates GAPDH inhibition with the induction of apoptosis.

[0141] The ratio of the expression of Bax and Bcl-2 is under the control of the transcription factor NFkB (NFκB). NFkB supports the survival of tumor cells. It exerts an anti-apoptotic effect by increasing the expression of Bcl-2 and prevents ROS by increasing the expression of antioxidant enzymes. The finding that 2250 induces a decrease in the expression of Bcl-2 and an increase in ROS supports the view that 2250 directly or indirectly inhibits NFkB.

[0142] Figure 5 shows the regulation of the expression of the cancer proteins Bax and Bcl-2. The effects of treatment with 200 μM GP-2250 over 0, 6, 12, and 24 hours on the expression of the cancer proteins a) Bax and b) Bcl-2 were tested in PancTuI cells by Western blot using α-tubulin as a control. While the expression of the pro-apoptotic protein Bax increased, the expression of the anti-apoptotic Bcl-2 decreased with the time of incubation with GP-2250.

[0143] Example 9 In an example of reducing the toxicity associated with chemotherapeutic drugs and synergistically increasing cytotoxicity, the following combinations of GP-2250 with the chemotherapeutic drugs gemcitabine, mitomycin C, and cisplatin were tested in patient-derived pancreatic cancer cell lines (Bo80) and two mesothelioma cell lines (JL-1, MSTO-211H).

[0144] (1)Synergistic effect of GP-2250 and gemcitabine: When tested in primary pancreatic cancer cells (Bo80), the combination of GP-2250 and gemcitabine at concentrations that were themselves inactive produced strong cytotoxicity when the drugs were combined at their respective inactive doses (200 μM of GP-2250 + 100 μM or 1000 μM of gemcitabine) (Figure 6). The reduction in chemotherapy drug-related toxicity can be achieved in vitro by using the combination with GP-2250 while maintaining high efficacy. Therefore, the combination of GP-2250 and gemcitabine was tested in a mouse model of patient-derived xenografts to evaluate the therapeutic potential of this drug combination in vivo (see Figures 12 - 15 and 18).

[0145] Figure 6 shows the synergistic effect of GP-2250 and gemcitabine. Cell viability was tested in a primary cell line (Bo80) derived from human pancreatic cancer. Cells were incubated at 37°C for 24 hours with GP-2250 (200 μM, 500 μM, 1000 μM) or gemcitabine (G; 100 μM, 1000 μM) alone or in combination with both drugs. The concentrations of GP-2250 (200 μM) and gemcitabine (100 μM or 1000 μM) were themselves inactive. A significant synergistic effect was observed when combined. The number of viable cells decreased by 70% - 75%. Cell viability was tested by colorimetric analysis using the MTT assay. The yellow MTT dye is converted to purple formazan by viable cells (M5655 from Sigma).

[0146] (2) Synergistic effects of GP-2250 with mitomycin C and cisplatin shown in mesothelioma cells JL-1 and MSTO-211H: GP-2250 has significant synergistic effects when combined with either mitomycin C or cisplatin (Figures 7A and 7B). Importantly, this synergistic effect was evident at drug doses that were themselves inactive. The cytotoxic effects of CisP and mitomycin C can be enhanced by up to 30% by GP-2250. A reduction in the toxicity associated with chemotherapeutic agents can be achieved by using this combination to achieve high efficacy and then reducing the dose of the chemotherapeutic agent.

[0147] Figures 7A and 7B show the synergistic effects of GP-2250 with mitomycin C or cisplatin in mesothelioma cell lines JL-1 and MSTO-211H. Figure 7A: When JL-1 cells were incubated at 37°C for 24 hours with GP-2250 (200 μM, 750 μM) or mitomycin C (MMC; 0.5 μM, 1.0 μM) alone or in combination of both drugs, a synergistic effect of cytotoxicity was observed with the combination at a concentration that was itself inactive (250 μM GP-2250 and 1.0 μM MMC). Figure 7B: When MSTO-211H cells were incubated at 37°C for 24 hours with GP-2250 (250 μM, 1000 μM) or cisplatin (CisP; 0.5 μM, 2.5 μM) alone or in combination of both drugs, a synergistic effect of cytotoxicity was observed with the combination at a concentration that was itself inactive (250 μM GP-2250 and 2.5 μM CisP). The number of viable cells decreased by approximately 25% with the combined treatment. Cell viability was tested by colorimetric analysis using the MTT assay. The yellow MTT dye is converted to purple formazan by viable cells (M5655 from Sigma).

[0148] Example 10 The secondary resistance to 2250 was tested, mimicking the repeated bolus administration of the drug over up to 8 weeks. Gemcitabine was used as the comparator drug. Potential secondary resistance was expected to manifest as a decrease in the ability of any drug to impair cell viability. The study was conducted using three pancreatic tumor cell lines, AsPC-1, PancTuI, and Bo80, the latter being a primary cell line derived from a pancreatic cancer patient. Cells were treated once a week with 2250 or gemcitabine at a dose that destroys 80% - 90% of the cells for 24 hours. Subsequently, the medium was changed and the cells were allowed to regrow for 6 days in the absence of the drug.

[0149] This once-weekly cycle of cell killing and regrowth was repeated for both drugs over 4, 6, and 8 weeks. Non-treated cultures at 4, 6, and 8 weeks were used as controls. At 2 hours, 24 hours, and 6 days at each treatment cycle over 4, 6, and 8 weeks, cell viability was tested by BrdU assay or MTT assay. Compared to the non-treated controls, GP-2250 did not show a decrease in cytotoxicity after 4 weeks (Figure 8A), 6 weeks (Figure 9), and 8 weeks (Figure 10A) in all three cell lines. Since the cytotoxicity was consistent with that of the non-treated controls, induction of secondary resistance by GP-2250 was not observed. Therefore, GP-2250 is expected to provide a sustained benefit in the long-term treatment of cancer patients. In contrast, partial secondary resistance was already evident after 4 weeks for gemcitabine, as shown by the decrease in cytotoxicity at all concentrations tested (Figure 8B). The decrease in gemcitabine cytotoxicity was confirmed at week 8 (Figure 10B).

[0150] Figures 8A and 8B are tests for secondary resistance. After four once-weekly cycles of cytotoxic treatment and regrowth (see text), the cytotoxicity of GP-2250 (Figure 8A) and gemcitabine (Figure 8B) was tested in the AsPC-1 pancreatic cancer cell line using the BrdU assay and MTT assay, respectively (light bars). The control corresponded to cells cultured for 4 weeks without drug treatment (dark bars). Since the cytotoxicity did not change after four once-weekly treatment cycles, there was no evidence of secondary resistance for GP-2250. In contrast, secondary resistance occurred for gemcitabine, as indicated by the decrease in cytotoxicity after four once-weekly treatment cycles.

[0151] Figure 9 shows the results of the test for secondary resistance. After six once-weekly cycles of cytotoxic treatment and regrowth (see text), the cytotoxicity of GP-2250 was tested in the PancTuI pancreatic cancer cell line using the BrdU assay (light bars). The control corresponded to cells cultured for 4 weeks without drug treatment (dark bars). Since the cytotoxicity did not change after six once-weekly treatment cycles, there was no evidence of secondary resistance for GP-2250.

[0152] Figures 10A and 10B are tests for secondary resistance. After eight once-weekly cycles of cytotoxic treatment and regrowth (see text), the cytotoxicity of GP-2250 (Figure 10A) and gemcitabine (Figure 10B) was tested in the Bo80 primary pancreatic cancer cell line using the BrdU assay (light bars). The control corresponded to cells cultured for 8 weeks without drug treatment (dark bars). Since the cytotoxicity did not change after eight once-weekly treatment cycles, there was no evidence of secondary resistance for GP-2250. In contrast, partial secondary resistance occurred for gemcitabine, as indicated by the decrease in cytotoxicity after eight once-weekly treatment cycles.

[0153] Example 11 A mouse model of patient-derived xenografts (PDX) from combination therapy was created and tested as shown in FIGS. 11 to 15 and FIG. 18. For all therapies and controls in the PDX model experiments, they were administered intraperitoneally. Pancreatic cancerous tissue was transplanted into mice and allowed to grow to a specified volume of 200 mm 3 until it reached the specified volume.

[0154] FIG. 11 shows the relative tumor growth rates of patient-derived pancreatic tumor tissue (Bo122) for treatment with GP-2250 alone (squares) or nab-paclitaxel alone (dark triangles) compared to control treatment (circles), and treatment as combination therapy (light triangles). The combination treatment resulted in a partial regression of tumor volume. As shown for patient-derived pancreatic cancer tissue Bo122 in the PDX mouse model, in the combination group of 2250 (500 mg / kg * BW) and the standard agent nab-paclitaxel (15 mg / kg * BW), the tumor volume was characterized by partial regression. * p < 0.05.

[0155] FIG. 12 shows the relative tumor growth rates of patient-derived pancreatic tumor tissue Bo80 for treatment with GP-2250 alone (squares) or gemcitabine alone (dark triangles) compared to control treatment (circles) in the PDX mouse model, and treatment as combination therapy (light triangles). The combination treatment resulted in significant tumor regression.

[0156] FIG. 12: In the combination group of 2250 (500 mg / kg BW) and the standard agent gemcitabine (50 mg / kg), as shown for patient-derived pancreatic cancer tissue Bo80 in the PDX mouse model, a significant regression of relative tumor volume was observed when the combination was used. Data ± SEM. *** p < 0.001.

[0157] Figure 13: In the combination group of 2250 (500 mg / kg BW) and the standard agent gemcitabine (50 mg / kg), as shown in the PDX mouse model of patient-derived pancreatic cancer tissue Bo103, significant regression of relative pancreatic tumor volume was observed when the combination (light triangle) was used. The control is represented by a circle, and gemcitabine monotherapy is represented by a dark triangle. Tumor growth resumed after a 10-day treatment interruption but decreased again after resumption of treatment around day 70. Data ± SEM. *** p < 0.001.

[0158] Figure 14: 2250 (500 mg / kg * BW) and the standard agent gemcitabine (50 mg / kg * BW), in the combination group, pancreatic tumor growth was characterized by partial remission. The control is represented by a circle, 2250 is represented by a square, and gemcitabine monotherapy is represented by a dark triangle. As shown for patient-derived pancreatic cancer tissue Bo69 in the PDX mouse model, significant reduction in relative tumor volume was observed when the combination was used. Data ± SEM. *** p < 0.001.

[0159] Figure 15 shows the relative Bo70 pancreatic tumor growth rate for treatment with GP-2250 monotherapy (square) or gemcitabine monotherapy (dark triangle), and treatment as combination therapy (light triangle), compared to control treatment (diamond) in the PDX mouse model of patient-derived pancreatic cancer tissue.

[0160] Figure 15: 2250 (500 mg / kg * BW) and the standard agent gemcitabine (50 mg / kg * BW), in the combination group, for patient-derived pancreatic cancer tissue Bo70 in the PDX mouse model, tumor growth was characterized by disease stabilization. Data ± SEM. *** p < 0.001.

[0161] Example 12 Neuroendocrine tumors were tested in vitro using the neuroendocrine cell line QGP-1 and in vivo using a mouse model with QGP-1 cell xenografts and patient-derived pancreatic cancer tissue xenografts Bo99. Figure 16A shows the relative QGP-1 tumor cell viability in vitro for treatment with GP-2250 alone (light gray) or gemcitabine alone (dark gray) relative to the control. Figure 16B shows the synergistic effect of the combination therapy.

[0162] GP-2250 and gemcitabine each showed a concentration-dependent cytotoxic effect in QGP-1 cells (Figure 16A). The combination of both substances had a synergistic effect at concentrations of 175 μM GP-2250 and gemcitabine (0.01 μM) and 200 μM GP-2250 and gemcitabine (0.001 μM and 0.01 μM) (Figure 16B).

[0163] Figure 17 shows the relative QGP-1 cell tumor growth rates for treatment with GP-2250 alone (squares), gemcitabine alone (dark triangles), or treatment as combination therapy (light triangles) compared to control treatment (circles) in the mouse xenograft model.

[0164] From Figure 17, it is shown that the tumor growth in the combination group of GP-2250 (500 mg / kg * BW) and gemcitabine (50 mg / kg * BW) was characterized by a partial remission, and as shown for QGP-1 cells in the xenograft mouse model, a significant partial regression of the relative tumor volume was observed when the combination was used. Data ± SD. *** p < 0.001.

[0165] Figure 18 shows the relative Bo99 tumor growth rate of patient-derived neuroendocrine tumors (Bo99) for treatment with gemcitabine alone (dark triangles) and combination therapy (light triangles) compared to control treatment (circles) in the PDX mouse model. The combination treatment resulted in tumor volume regression. In the combination group of 2250 (500 mg / kg BW) and the standard agent gemcitabine (50 mg / kg), significant regression of the relative neuroendocrine tumor volume was observed in the mouse PDX model when the combination was used. Tumor growth resumed after a 10-day treatment interruption but decreased again after resumption of treatment around day 74. 2250 was only tested in combination. Data ± SEM.

[0166] Example 13 Chemotherapy-resistant stem cells were collected from human patients with stage 3 and 4 pancreatic cancer, which is advanced, and grown to obtain a larger population of stem cells. The chemotherapy-resistant stem cells were then exposed to several concentrations of gemcitabine alone, GP-2250 alone, and a combination of gemcitabine + GP-2250. As shown in Figure 19, gemcitabine alone showed a minimal effect at all concentrations tested, and GP-2250 alone had some effect at higher concentrations. However, the combination of gemcitabine and GP-2250 resulted in very significant cytotoxicity against the stem cells.

[0167] Although the subject matter of the present disclosure has been described and illustrated in considerable detail with reference to several exemplary embodiments including various combinations and sub-combinations of features, those skilled in the art will readily understand that other aspects as well as variations and modifications thereof are intended to be encompassed within the scope of the present disclosure. Further, the description of such aspects, combinations, and sub-combinations is not intended to imply that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of the present disclosure is intended to include all variations and modifications that fall within the spirit and scope of the appended claims.

Claims

1. Use of compound 2250 for the manufacture of a composition for treating neuroendocrine tumors by inhibiting GAPDH activity in a subject, 【Chemical 1】 wherein the composition comprises compound 2250, a pharmaceutically acceptable salt, hydrate, or solvate thereof, or a combination thereof, and the composition is administered to the subject.

2. The use according to claim 1, wherein the composition is administered intravenously, subcutaneously, intramuscularly, topically, orally, intraperitoneally, intrathecally, intranasally, transocularly, intrapulmonary, transdermally, intravitreally, by inhalation, tracheally, by intravitreal injection, or a combination thereof.

3. Use of compound 2250 and mitomycin in the manufacture of a kit for treating mesothelioma by inhibiting GAPDH activity.

4. Use of compound 2250 and cisplatin in the manufacture of a kit for treating mesothelioma by inhibiting GAPDH activity.

5. Use of compound 2250 and paclitaxel in the manufacture of a kit for treating pancreatic cancer by inhibiting GAPDH activity.

6. Use of compound 2250 and gemcitabine in the manufacture of a kit for treating neuroendocrine tumors by inhibiting GAPDH activity.

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