Polymeric iron-binding compositions and methods of use for treating disorders associated with excess injury reactive iron - Patents.com

Abstract

Disclosed herein is an iron chelating polymer for the treatment of a disease involving an iron-mediated pathology, the polymer comprising the reaction product of a first monomeric unit and a second monomeric unit polymerized by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent, the first monomeric unit being selected from the group consisting of compound (I): [Formula 1] Represented by TIFF2025510226000031.tif42128, The second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole and styrene, the iron chelating polymer is dissolved in an aqueous medium, the iron chelating polymer has a molecular weight of at least about 1500 Da prior to chelation, and the iron chelating polymer includes one or more intramolecular hexadentate ligands for chelating iron. The described polymers are useful for the treatment of diseases associated with excess reactive iron.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 323,304, filed March 24, 2022, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods and uses of iron chelation, and more particularly, to methods and uses for the treatment of diseases associated with excessive and damaging reactive iron. [Background technology]

[0003] Iron as a biological catalyst and iron overload toxicity. Iron is an essential trace nutrient metal used by all vertebrates, as it provides the active catalytic center for various enzymes involved in DNA synthesis, metabolism and cell defense, and, with few exceptions, cannot be replaced by alternative trace metals. Approximately 2% of human genes code for iron proteins, more than half of these have catalytic function, with the largest proportion being enzymes of the oxidoreductase class (Andreini et al., 2018).

[0004] Table 1 provides examples of iron-dependent enzymes that are required by most cells for many important physiological functions. These enzymes are involved in cell growth and replication, and iron generally cannot be replaced while retaining enzymatic activity.

[0005] [Table 1]

[0006] However, iron atoms can be toxic due to chemical reactions catalyzed by free reactive iron atoms that can generate reactive oxygen radical species (ROS) in aerobic systems at physiological pH values. These ROS chemical oxidizing radicals can damage cells and tissues, leading to cell and tissue damage and disease.

[0007] Free reactive iron atoms can generate ROS by using superoxide (O 2 - ) is Fe 3+ Fe 2+ of electrons with simultaneous reduction to O 2 The human body consumes approximately 5 g of O per day. 2 - (Hayyan et al., 2016), but superoxide dismutase (an iron-dependent enzyme) neutralizes the above-normal amounts normally required for cell regulation and signaling (Ray et al., 2012). Superoxide is also produced by nicotinamide adenine phosphate oxidase in vertebrate phagocytic defense cells and can be used to kill phagocytosed invading microorganisms (Minakami et al., 2006). Superoxide can set up a cascade cycling of Fe reduction / oxidation coupled to peroxide and hydroxyl radical ·OH generation, as shown below, which is highly toxic via damage to DNA and membrane lipids (Halliwell et al., 2021).

[0008] [ka]

[0009] Thus, regulation of the body's iron supply is important to ensure sufficient amounts of iron for needs, and also to prevent accumulation of excess amounts of iron above normal levels. As explained in more detail herein, vertebrates generally achieve this iron homeostasis by controlling the amount of iron taken up from food in the intestine, by delivering iron onto transferrin, a chaperone carrier protein that substantially prevents the iron from reacting while it is delivered to cells for use, and by maintaining sufficient iron reserves in ferritin intracellular stores where the stored iron is substantially shielded, both spatially and chemically, and non-reactive.

[0010] Furthermore, because iron is also required by pathogens to invade and infect the body, there is an active iron-suppressing defense that occurs early during infection involving a reduction in the amount of circulating iron on transferrin, limiting access to iron required by pathogens for proliferation. For a review of infection-related aspects, see Holbein et al. (2021), the entire contents of which are incorporated herein by reference.

[0011] Chemical containment and regulation of normal iron homeostasis Containment of reactive iron and its ROS products can be achieved both by compartmentalization and by its chemical chaperoning. Phagocytes, including macrophages and polymorphonuclear leukocytes, can compartmentalize ROS generation within intracellular phagosomes that protect other intracellular organelles and the extracellular environment from direct ROS exposure (Fang, 2004). The majority of whole-body iron stores, equivalent to approximately 55 mg / kg, are located intracellularly and incorporated into hemoglobin (red blood cells), myoglobin (muscle cells), and ferritin (hepatocytes) (Yiannikourides et al., 2019). Iron incorporated into heme and ferritin may not be freely chemically available to participate in ROS generation. Body iron stores are transported to sites of use in other cells by transferrin, which binds iron, and iron is chaperoned in the circulation and unavailable for ROS reactions. There are additional small labile iron pools located both intracellularly and extracellularly that are readily ROS-reactive (Kakhlon et al., 2002), and the pools are associated with iron dysregulation that can increase the amount in the labile pool.

[0012] Several currently used therapeutic agents, including aminoglycoside antibiotics, can bind iron that does not fully meet the chaperonin requirements (Ezraty et al., 2016) and induce ROS-associated tissue damage, likely due to mobilization of labile iron (Li et al., 2009). As a result, the need for containment and chaperoning of labile ROS-reactive iron in the host as part of normal homeostatic regulation is highlighted.

[0013] Of the 3750 mg of total iron in the human body, approximately 65% ​​is incorporated into heme in red blood cells, 10% into heme in myoglobin in muscles, 14% into macrophage cells of the reticuloendothelial system (RES), 28% is stored within ferritin in hepatocytes, and 4% is stored in bone marrow cells (Yiannikourides et al., 2019). Iron taken up by enterocytes in the intestine can be temporarily stored in small amounts in ferritin until there is a demand for iron replacement elsewhere in the body, after which transferrin can take up the iron into the bloodstream acting as a shuttle / delivery protein chaperone. The transferrin pool is normally only 30% saturated with iron and thus can provide an iron retention capacity that limits the amount of labile non-transferrin-bound iron in the circulation. The transferrin iron pool represents a flux of approximately 25 mg of iron per day (Pantopoulos, 2018). The body has no mechanism for excreting iron, and small losses of 1-2 mg per day from skin desquamation and other sources are correspondingly replaced by intake from the intestine.

[0014] Hepcidin, a 25 amino acid peptide hormone produced primarily by liver hepatocytes, is a regulator of iron homeostasis and can negatively regulate iron uptake by inactivating plasma membrane-bound ferroportin on enterocyte and macrophage cell membranes as required for the movement of intracellular iron to the extracellular compartment (Nemeth et al., 2022). As a result, tight regulation of iron homeostasis is provided, involved in steps that ensure there are no excessive toxic amounts of iron either intracellularly or extracellularly. Iron dysregulation often indicates elevated amounts of circulating plasma iron, including >50% saturation of transferrin-bound iron, along with elevated non-transferrin-bound iron (NTBI) that can reach >0.5 μM (Vinchi, 2021). The labile portion of NTBI can play a role in the pathogenesis of various nonmicrobial diseases discussed below, but elevated plasma iron in the form of transferrin-bound iron can also support microbial infections (Holbein et al., 2021).

[0015] Underlying iron dysregulation disorder Nature of iron dysregulation Iron dysregulation disorders can result from an increased amount of circulating labile reactive plasma iron. This can be evidenced by elevated plasma transferrin-bound iron (TBI), with transferrin saturation exceeding normal 30% and sometimes reaching 100% saturation (Akinc et al., 2011). High transferrin saturation can result in a portion of plasma iron not being effectively chaperoned, providing a pool of non-transferrin-bound iron (NTBI) labile reactive iron (LPI). This LPI is furthermore more mobile and can enter cells where it is toxic (Cabantchik et al., 2005; Cabantchik, 2014).

[0016] Although it is now well established that iron dependence supports the growth of microbial pathogens or cancer cells due to an increased need for iron, there is growing evidence that dysregulation of normal iron homeostasis in vertebrates is involved in the pathogenesis of other diseases, with more than normal amounts of unprotected / unchaperoned and chemically reactive free iron in some iron pools and compartments of the body. Diseases with iron dysregulation may not be directly related to the need for iron for the growth of pathogenic microorganisms or cancer cells.

[0017] Excess plasma iron in the form of TBI and NTBI LPI can affect two major categories of disease. For example, elevated plasma iron in the form of transferrin-iron can support infections and cell proliferative disorders, including cancer. Increased plasma iron present as non-transferrin-bound LPI can be part of the pathology of other diseases in which excess reactive iron causes iron-related pathology.

[0018] Examples of various diseases from both categories are summarized in Tables 2 and 3 below.

[0019] [Table 2]

[0020] [Table 3]

[0021] A brief overview of various diseases associated with iron dysregulation is provided below.

[0022] Infection: All pathogenic microorganisms, except Borrelia burgdorferi, the causative agent of Lyme disease, require iron for growth and replication in humans (Schaible et al., 2004). Borrelia burgdorferi can utilize manganese instead of iron (Troxell et al., 2013). The vertebrate host normally maintains a state of low iron bioavailability to microorganisms, including extracellular compartments such as plasma, respiratory secretions, and tears, where infections can be initiated, providing natural nutritional immunity. Transferrin in plasma and lactoferrin in tears and other secretions can maintain very low levels of freely available iron (Murdoch et al., 2022). Lactoferrin concentrations can reach >3 mg / mL in tears (Hanstock et al., 2019). Furthermore, when infection is first detected, the body can mount an initial iron withdrawal defense response in which extracellular iron concentrations are further reduced by moving this iron to intracellular stores (Nemeth et al., 2022). Successful pathogens can deploy various virulence mechanisms that compete for iron, as reviewed elsewhere ( Holbein et al., 2021 ).

[0023] Pathogenic microorganisms: Iron has been found to support infection by several pathogenic microorganisms, and Table 4 summarizes several microbial infections in relation to the primary host iron source that imparts infection. Some of these have cell wall surface receptors that can interfere with the host iron source, while others can deploy high affinity siderophores to strip iron from host transferrin or lactoferrin (Holbein et al., 2021).

[0024] [Table 4]

[0025] Bacterial sepsis: Sepsis is a life-threatening condition that causes approximately 110,000 deaths worldwide each year (Olwal et al., 2021). It can result from severe dysregulation of the host response to inflammation. Sepsis can arise from viral infections such as COVID-19 (Olwal et al., 2021) and also through microbial infections. Bacterial sepsis can develop from severe bacterial infections with a stimulated and dysregulated host inflammatory response (Liu et al., 2021) and is often caused by bacterial cell components released during infection, including endotoxins (Lehmann et al., 2015).

[0026] During sepsis, iron metabolism may be altered with increased iron uptake into cells and has been associated with increased iron-driven oxidative damage and cell death (Liu et al., 2021). High serum iron levels are further associated with sepsis and poor sepsis outcomes (Lan et al., 2018). Transferrin saturation, which reflects serum iron availability, correlates with sepsis outcomes, with increased iron availability being prominent in subjects with lethal illness (Liu et al., 2021; Tacke et al., 2016). This aspect driven by excess iron is in addition to iron-supplying microbial infections, as it is associated with dysregulated inflammation of the host. Thus, a state of iron dysregulation may provide a state of increased iron availability to support rapidly growing microorganisms on the one hand, and increased host damage from the associated dysregulated inflammatory response on the other hand.

[0027] Parasitic pathogens: Multicellular parasites may further require iron to invade and initiate infection in vertebrate hosts (Mach et al., 2020). Leishmania chagasi, which causes leishmaniasis, can utilize iron from transferrin, lactoferrin, or heme when growing in a promastigote form and may directly uptake iron without deploying an iron-blocking siderophore (Wilson et al., 1994). Trypanosoma brucei can use the transferrin surface receptor and Entamoeba histolytica can use the lactoferrin receptor for iron acquisition (Mach et al., 2020). Plasmodium falciparum, which causes malaria, has also been shown to have a surface receptor for transferrin-iron present within hemoglobin-rich red blood cells (Clark et al., 2014). Iron supplementation has been shown to promote infection with Trypanosoma cruzi (Lalonde et al., 1984), and iron supplementation has been associated with an increased risk of malaria (Clark et al., 2014). Parasitic infections, in contrast to most microbial infections, can be chronic in nature and are associated with what is described as anemia of chronic disease (Wicinski et al., 2020). This can also occur in cancer, where this chronic anemia can be considered the result of a prolonged effort by the host to restrict iron to invading pathogens or cancer cells. Host iron deficiency has been found to be protective against malaria based on epidemiological studies (Clark et al., 2014).

[0028] Viral infection: Viral replication in host cells can be influenced by host iron in the case of both DNA viruses, such as Hepatitis B (HBV) and human cytomegalovirus (HCMV), and RNA viruses, such as Hepatitis C (HCV) and human immunodeficiency virus (HIV) (Schmidt, 2020). It remains unclear whether iron dysregulation predisposes the host to viral infections, or whether viral infections cause host iron dysregulation. Elevated iron levels are associated with the progression of chronic HBV infection (Wei et al., 2018), and iron supplementation has been shown to enhance HCV viral replication in vitro (Kakizaki et al., 2000). Transferrin receptor-1 mRNA levels increased due to HIV infection, resulting in increased iron uptake and higher levels of cellular iron (Chang et al., 2015). However, it has been suggested that in cirrhotic subjects, HBV-associated liver injury, rather than HBV infection per se, may cause changes in serum iron markers ( Mao et al., 2015 ).

[0029] Cancer: The role of iron and iron dysregulation in cancer is multifaceted as reviewed by Torti et al., (2018). Stevens et al. (1994) reported on a cohort of 14,000 US National Health and Nutrition Examination Survey participants and found that participants with higher transferrin (Tf) iron saturation levels were at higher risk of cancer than those with lower transferrin Tf saturation levels, a finding supported by later studies (Wu et al., 2004). Cancer cells arising from various tissues such as breast or liver have an increased need for iron compared to normal cells and may increase the amount of cell surface transferrin receptor in response to the increased ongoing need for iron (Greenshields et al., 2019). Targeting the transferrin receptor has been proposed for therapeutic potential (Shen et al., 2018), as shown using an antibody against the cancer cell surface transferrin receptor TfR1 (Candelaria et al., 2021). This aspect is appealing because common features of all cancers are the possession of the TfR1 receptor (Candelaria et al., 2021), altered iron metabolism and increased need for iron to support rapid proliferation (Zhang et al., 2020), and a primary dependency on host transferrin as a source of this iron. Any advances in beneficial iron restriction can therefore be broadly applicable to a variety of cancers.

[0030] Cancer can further induce chronic anemia, as seen in other chronic diseases, likely a host attempt to limit cancer growth (Greenshields et al., 2019).Somewhat paradoxically, cancer cells are more susceptible to ferroptosis, a programmed cell death triggered by excess reactive iron (Lei et al., 2019), suggesting that host iron withdrawal defenses that limit cancer growth may also impede cancer cell killing via ferroptosis.

[0031] Ferroptosis cell death: Ferroptosis is a further form of regulated cell death (RCD), but unlike other forms such as apoptosis and necrosis, it can be triggered by iron-dependent accumulation of lipid peroxides that kill cells, but shares other common features with other modes of RCD (Lei et al., 2019). ROS produced via the iron-catalyzed Fenton reaction may contribute to its initiation (Toyokuni et al., 2020), and iron dysregulation due to excess labile iron may be a predisposing factor.

[0032] Potential inhibitors of ferroptosis, including ROS-scavenging antioxidants such as α-tocopherol and ferrostatin-1, have been investigated and have shown potential to reduce ferroptosis damage ( Angeli et al., 2017 ).

[0033] Excess labile iron driving ferroptosis has also been demonstrated through inhibition by the addition of the iron chelator deferoxamine (Yang et al., 2008). However, iron-nitriloacetate (NTA) has been shown to promote ferroptosis and kidney cancer by driving Fenton activity (Toyokuni et al., 2020). Various iron chelators may differ in their ability to fully coordinate and therefore chaperone iron (Holbein et al., 2021). Deferoxamine can fully hexadentate iron within a single deferoxamine molecule, whereas NTA requires two chelator molecules to fully fill a single iron atom. Thus, depending on the prevailing chelator and iron concentration, poorly coordinated Fenton reactive species may form and appear to underlie the toxicity of some of the currently used medical chelators (Holbein et al., 2021). Furthermore, small molecule cell-permeable chelators can more easily penetrate cells to reach cellular iron stores and mobilize additional labile iron supplies, which in turn may exacerbate iron dysregulation.

[0034] The oversupply of labile reactive iron observed as a hallmark of iron dysregulation may underpin tissue toxicity in many diseases, and ferroptosis appears to be at least part of the disease pathology, as discussed further below.

[0035] Inflammatory diseases: Although inflammation typically accompanies infections and cancer, other diseases appear to be primarily inflammatory in nature, and iron dysregulation and associated ROS activity may be involved in the inflammatory response.

[0036] Eye: Ocular iron dysregulation has been linked to various eye diseases affecting the cornea and retina, including corneal epithelial damage, corneal endothelial cell dysfunction, retinal pigment epithelium (RPE)-associated eye diseases, glaucoma, diabetic retinopathy (DR), retinal ischemia / reperfusion injury (RIRI), retinoblastoma, retinitis pigmentosa (RP), and age-related cataract, which commonly involve ferroptotic pathology (Zhang et al., 2022). Ferroptotic iron-induced toxicity through ROS damage may be associated with corneal diseases such as cataractogenesis, inflammatory retinal diseases such as age-related macular degeneration (AMD), and optic neuropathy (Loh et al., 2009). Transferrin mRNA levels may be upregulated in AMD, possibly in response to increased retinal iron load (Chowers et al., 2006). This feature may provide a potential avenue for new therapeutics, including the use of iron chelators, to treat ocular diseases for which new therapeutics are urgently needed.

[0037] Lung iron content is regulated as excess iron can catalyze ROS formation and is associated with the pathogenesis of chronic inflammatory lung diseases such as idiopathic pulmonary fibrosis (Ogger et al., 2020). Iron accumulation is increased in lung sections from subjects with IPF, and human lung fibroblasts exhibit greater proliferation as well as cytokine and extracellular matrix responses when exposed to increased iron levels (Ali et al., 2020). The authors provided direct evidence of iron overload affecting the progression of pulmonary fibrosis. They investigated whether alterations in iron homeostasis are a cause or consequence of pulmonary fibrosis and, using a mouse model of iron overload, showed that iron accumulation leads to lung dysfunction and subsequently exacerbates pulmonary fibrosis upon bleomycin-induced lung injury (Ali et al., 2020). Pulmonary fibrosis is a progressive, irreversible disease as fibrotic lung tissue does not repair / remodel, urgently requiring antifibrotic agents. Overcoming iron dysregulation to reduce iron-driven fibrosis is a potential new avenue for treatment. Iron chelating therapeutics have potential for use in slowing or halting pulmonary fibrosis.

[0038] Kidney: Increased renal proximal tubule (PT) cell cytosolic non-transferrin Tf binding, i.e., labile iron, has been shown to induce the generation of ROS in PT cells and may contribute to the progression of proteinuric chronic kidney disease (Smith et al., 2009). Recent clinical studies using antioxidants capable of reducing labile iron suggest that iron chelation in the kidney has beneficial effects on the course of chronic kidney disease (Swaminathan et al., 2008). In nephrotic syndrome, associated with damage to the glomerular filter, urinary Tf concentrations may be increased and may even cause hypotransferrinemia, iron loss, and microcytic anemia (Prinsen et al., 2001).

[0039] Nephritic kidney damage accompanied by urinary transferrin excretion is also part of the pathology associated with autoimmune diseases, including lupus erythematosus (Theut et al., 2020) and rheumatoid arthritis (Kochi et al., 2018), as discussed further below.

[0040] Diabetes: High dietary iron and abnormal iron metabolism may be risk factors for type 2 diabetes mellitus (T2DM), influencing most of its features: reduced insulin secretion, insulin resistance, and increased hepatic gluconeogenesis (Harrison et al., 2023). Dysregulation of iron metabolism with increased serum levels of ferritin is found in newly diagnosed type 2 diabetes, but not in individuals with prediabetes (Venkatesan et al., 2021). Oxidative stress from ROS is now known to be an underlying mediator of diabetic complications (Giacco et al., 2010). Iron and ferroptosis have been shown to be involved in pancreatic β-cell death (Li J. et al., 2020). In a model study with T2DM, insulin secretion in mice was exacerbated by ferroptosis-inducing compounds. However, quercetin (a natural iron chelator), the ferroptosis inhibitor ferrostatin-1, and the iron chelator deferoxamine each restored cell viability when cells were treated with high glucose (Li D. et al., 2020). These studies support the potential use of iron chelating therapeutics in the treatment of T2DM.

[0041] Cardiovascular: Ferroptosis driven by iron dysregulation has been implicated in several cardiovascular disease conditions, including cardiomyopathy, atherosclerotic disease, and myocardial ischemia / perfusion injury (Li et al., 2021; Fang et al., 2023). Inhibition of ferroptosis by ferrostatin-1 improved cardiac function and reduced mortality in a doxorubicin-induced mouse model of cardiomyopathy, and was associated with the release of free cellular iron triggered by HO-1 upregulation (Fang et al., 2019). However, the mechanisms of ferroptosis in cardiac and vascular diseases remain unclear (Li et al., 2021).

[0042] Autoimmunity: The role of iron regulation in immune-related diseases was recently reviewed by Cronin et al. (2019). Considerable evidence now links iron dysregulation to the pathogenesis of lupus erythematosus (Wincup et al., 2021). Lupus nephritis is associated with renal iron accumulation (Theut et al., 2020) and ferroptosis, a renal cell injury, has been described as a key feature of the pathology (Wincup et al., 2021).

[0043] Nervous system: Many nervous system conditions have been shown to coincide with altered somatic distribution of various transition series biometals, especially in the case of iron (Pfaender et al., 2014). Iron dysregulation with increased and unstable plasma iron supply and resulting increased ROS causes oxidative stress and damage to neural tissue and is now linked to Friedreich's ataxia, Alzheimer's disease (AD), Parkinson's disease, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) (David et al., 2022). Increased ferroptosis with corresponding destruction of neuronal cells has been reported in Parkinson's disease and MS (Hadzhieva et al., 2014) as well as for Alzheimer's disease (Das et al., 2021).

[0044] For example, Parkinson's disease (PD) can be characterized by a progressive motor disorder resulting from the progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta. In addition to iron accumulation, increased production of reactive oxygen / nitrogen species (ROS / RNS) and increased inflammatory markers can also be seen in this condition (Medeiros et al., 2016). Furthermore, abnormal iron increases can be detected in AD subjects, although controversy remains regarding the association of iron with AD plaques (Das et al., 2021). A common feature of iron dysregulation underlying neurological diseases appears to be iron-driven ferroptotic neuronal cell death (Ren et al., 2020).

[0045] Iron overload: While body iron overload can occur after repeated blood transfusions, as seen in subjects with thalassemia, i.e. because the body lacks an excretion pathway for excess iron, congenital disorders of iron overload, such as hemochromatosis, may represent iron overload disorders caused by dysregulation of iron homeostasis. In hemochromatosis, disruption of the hepcidin pathway due to mutations in genes encoding cofactors in iron signaling to hepcidin may result in insufficient hepcidin response to iron intake or high body iron stores. This may cause loss of hepcidin-mediated feedback inhibition in dietary iron absorption, resulting in unregulated intake of dietary iron, elevated transferrin iron saturation, and the appearance of labile reactive non-transferrin-bound iron (Pantopoulos, 2018).

[0046] Cirrhosis: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease that begins with the presence of excess lipid accumulation >5% in the liver and typically progresses to nonalcoholic steatohepatitis, fibrosis, cirrhosis, and often hepatocellular carcinoma (Chen, 2022). Excess free reactive iron and its associated ROS-mediated tissue damage are associated with the severity of NAFLD (Chen, 2022).

[0047] Anemia of chronic infection and inflammation: This anemia, called anemia of inflammation (AI) or often anemia of chronic disease (ACD), is a secondary anemia that can develop gradually as a result of chronic inflammation in chronic infections (especially parasitic infections), cancer, and at least other inflammatory diseases such as diabetes and autoimmune diseases (Wicinski et al., 2020; Ismaiel et al., 2020; Weiss et al., 2019). Overall, it appears to be a host defense mechanism. A short-term hypoferremic response is often seen early in the acute phase of infection and has been shown to be triggered by inflammatory mediators such as ILK-6, demonstrating a relatively short-term active mechanism to limit iron supply to the proliferating invader (Holbein et al., 2021). Identification of subjects with true iron deficiency may present a diagnostic challenge in AI / ACD. Subjects may require specific assessment of the source of blood loss and iron-targeted management strategies (Weiss et al., 2019).

[0048] Treatment options Iron restriction Higher heme iron intake and increased body iron stores were associated with a higher risk of type 2 diabetes, as shown in a meta-analysis of 11 prospective studies (Bao et al., 2012). Phlebotomy to reduce total body iron stores has shown potential benefit in the treatment of diabetes (Fernandez-Real et al., 2002).

[0049] In principle, restricting iron intake can lower the amount of harmful, unstable reactive iron. However, iron suppression (or supply) has remained controversial in medicine for many years due to the delicate balance required for iron homeostasis and the consequences of causing anemia. Although dietary iron supply would be safer, administration of parenteral iron to treat anemia, especially anemia of chronic disease, has significant consequences. Considering the role of dysregulated iron in infectious and other diseases, it requires careful consideration and should probably be avoided if possible.

[0050] Iron chelators Compositions that chelate and inhibit iron in the extracellular environment of proliferating cells have been previously disclosed to limit the uptake of iron required for use in intracellular proliferation and to affect cell proliferation. These disclosures can be found, for example, in U.S. Patent No. 10,709,784, entitled "Metal chelating compositions and methods for controlling the growth or activities of a living cell or organism," and U.S. Patent No. 11,059,785, entitled "Polymeric metal chelating compositions and methods for controlling growth and activities of living cells," both of which are incorporated herein by reference in their entirety. Such compositions have been shown in various examples, as reviewed by Holbein et al. (2021), to aid in the natural iron withdrawal defenses of vertebrates to combat diseases in which iron supply in the body promotes the proliferation of pathogens (i.e., either microbial invading pathogens or cancer cells with uncontrolled proliferation occurring within the animal's body).

[0051] These various disclosures referenced above show that application of iron chelating compositions that are soluble in aqueous media and have a molecular weight large enough (i.e., nominally about >1500 Da) so as not to be internalized normally by living cells, but capable of binding iron in the environment of living pathogenic cells, can withhold the supply of essential iron required for the proliferation of the pathogenic cells in their intracellular aspects. Thus, these compositions have been shown to address diseases in animals where iron supply plays a vital role in the growth and pathogenesis of invading pathogenic microorganisms or rapidly proliferating pathogenic cancer cells arising from within the body.

[0052] These disclosures further show that low molecular weight iron chelators with molecular weights of about <1500 Da, such as those currently in clinical use to treat iron overload diseases in humans (e.g., thalassemia) and that can more easily penetrate animal cells, are not sufficiently effective in limiting the growth of pathogenic microorganisms or cancer cells and often exhibit unacceptable toxicity to the treated animal. Table 5, adapted from Holbein et al. (2021), summarizes the properties and contrasts currently clinically approved chelators for use in humans as well as the proposed clinical chelator SP-420, compared to DIBI. DIBI is an example of a composition prepared as disclosed in U.S. Pat. Nos. 10,709,784 and 11,059,785. The chemical structures of these chelators are shown below.

[0053] [ka]

[0054] [Table 5] a Gumbau-Brisa et al.,2020;b Vlachodimitropoulou et al.,2017;c Holbein,2018;d Foley & Simeonov,2012;e Parquet et al.,2018;f Fokam,et al.,2020;g Holbein et al.,2021;h Zeidan & Griffiths,2018;i Badeli et al. al.,2019;j Holbein,2018;k Thompson et al.,2012;l Neupane & Kim,2010;m Luo et al.,2014;n Savage et al.,2018;o Parquet et al.,2019;p Ibrahim et al.,2010;q Bergeron,Raymond J.,et al.2014;r Hider,Robert C.,et al.2015;s Taher,Ali T.,et al.2017.

[0055] Liu et al (2010) (US Pat. No. 8,334,320) disclosed polystyrene nanoparticles with iron chelator moieties (e.g., deferiprone or deferoxamine) covalently bound to the surface of the nanoparticles for use in the treatment of osteoblasts to limit oxidative damage, and for the potential treatment of Parkinson's, Alzheimer's and Friedreich's ataxia diseases. However, the disclosed nanoparticles are insoluble, and no successful treatments have been reported from such insoluble compositions. Polymeric insoluble compositions may not be effective for administration to humans or other animals, and may not be able to reach the immediate environment surrounding diseased cells, i.e., to address excess iron in the immediate environment or within diseased cells.

[0056] Attempts to use small molecule iron chelators such as those reviewed by Holbein et al. (2021) and described above, including newer experimental chelators such as deferiprone for the treatment of experimental retinal disease (Ueda, K. et al., 2018) or FBS0701 for the treatment of diabetes (Cooksey et al., 2010), have generally not provided satisfactory results, which is in part related to the reported toxicity of the small molecule chelators used.

[0057] A newer small molecule chelator, SP-420, structurally related to FBS701 (see above) and developed to overcome the nephrotoxicity and other limitations of FBS701 and other similar molecules (Bergeron et al., 2014), still shows undesirable nephrotoxicity in clinical trials (Taher et al., 2017). Such toxicity issues can severely limit the dosage, route of administration to the body, and frequency of administration, and as a result, these limitations severely impair therapeutic efficacy.

[0058] Furthermore, iron chelators themselves, or iron chelators derived from microbial siderophores (i.e., with respect to their constituent iron-binding sites, such as SP-420, derived from the siderophore desferrithiocin), may undesirably promote microbial infections when used as iron chelators to treat iron-related diseases in humans. For example, this significant limitation was made evident through the use of deferoxamine, which is also a microbial siderophore, as a chelator to alleviate iron overload in humans (see Table 5).

[0059] Although several clinical trials of natural products with iron chelating properties, such as curcumin and polyphenols, have shown promise in lowering excess iron ( Xu et al., 2021 ), none have been approved for clinical use.

[0060] Hepcidin mimetics, stimulators of its production, and ferroportin inhibitors are undergoing early clinical stage testing for safety and potential efficacy (Casu et al., 2018). The ferroportin inhibitor VIT-2763 has progressed to Phase II trials and has shown both low toxicity and good potential to reduce serum iron levels (Taher et al., 2022). Other approaches using hepcidin mimetics, including PTG-300 and rusfertide, have shown promising Phase III results. Currently, none of these have received regulatory approval for ongoing clinical use (Verstovsek et al., 2021).

[0061] Although deferoxamine is capable of fully coordinating iron onto a single chelator molecule and is the only small molecule chelator currently in clinical use, its ability to support microbial growth and infection severely limits its use, as summarized in Table 5. Attempts to use deferoxamine in vivo have resulted in fatal infections in test animals (Kemp et al., 1995).

[0062] Regarding the reactivity of deferoxamine-bound iron, the stability of deferoxamine-bound iron has also been called into question by the finding that deferoxamine-starch conjugate compositions have been shown to induce oxidative damage to red blood cells in relation to the generation of ROS and causing cell and tissue damage (Niihara et al., 2000).

[0063] A need exists to address one or more of the deficiencies in the art as outlined above. Summary of the Invention

[0064] In one embodiment, the present disclosure provides a chelating composition soluble in an aqueous medium for chelating iron, the chelating composition comprising: A support material; one or more suitable types of iron-binding chemical groups fixed or incorporated into the structure of the support material; The one or more suitable types of iron-binding chemical groups are one or more of the following types: carboxyl, hydroxyl, phenolate, catecholate, hydroxamate, hydroxypyridinone and hydroxyphenyltriazole carboxyl; the carrier material comprises vinylpyrrolidone, imidazole acrylamide or styrene; The chelating composition has a minimum molecular weight large enough, nominally a molecular weight of about >1500 Da, so that it is not normally incorporated into intracellular aspects of the interior cell membrane of living animal cells, and is capable of binding iron with maximal complete chemical coordination of the bound iron on or within a single molecule of the composition; The chelating composition binds iron and remains substantially soluble with bound iron in the extracellular environment of the cell, and reduces uptake of iron into the interior intracellular aspects of the cell membrane of living animal cells; As a result, the external and internal aspects of the plasma membrane of living animal cells, as well as the internal aspects of the cell underlying the plasma membrane, are protected from chemically mediated damage such as that caused in whole or in part by excess, supranormal amounts of iron either in the external environment or within the plasma membrane of the animal cell.

[0065] In a further embodiment of the chelate composition, the composition comprises: and prepared from at least a first monomeric unit and a second monomeric unit, the first monomeric unit comprising a suitable metal binding chemical group incorporated or fixed thereto, optionally independently selected from the group consisting of carboxyl, hydroxyl, phenolate, catecholate, hydroxamate, hydroxypyridinone and hydroxyphenyltriazole; At this time, the first and second monomer units are polymerized by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent; Optionally, the first monomer unit is compound (I):

[0066] [ka] is represented by During the ceremony, R 1 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 2 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 3 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12; Optionally, the second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene.

[0067] In a further embodiment of the above chelate composition, the one or more suitable metal binding chemical groups is a hydroxypyridinone:

[0068] [ka] In the formula, X, Y and Z are independently N or C, When X is N and Y and Z are C, When Y is N and X and Z are C, When Z is N, X and Y are C.

[0069] In a further embodiment of the above chelate composition, compound (I) is compound (Ia):

[0070] [ka] It is prepared by polymerizing During the ceremony, R 1 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 2 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; PG is a protecting group, Compound (Ib)

[0071] [ka] is represented by During the ceremony, n is 1 to 12; R 4 COCCH 2 R 3 or a protecting group, Next, R 4 COCCH 2 R 3 removing PG to obtain compound (I); or R 4 When is a protecting group, R 4 and reacting with a suitable acrylate source to remove PG to give compound (I).

[0072] In a further embodiment of the above chelate composition, R 1 is H, R 2 is methyl, R 3 is methyl, n is 1 to 6, or optionally n is at least 2.

[0073] In a further embodiment of the above chelate composition, the first monomer unit is compound (II):

[0074] [ka] It is represented by:

[0075] In a further embodiment of the above chelate composition, the second monomer unit is 1-vinyl-2-pyrrolidone.

[0076] In a further embodiment of the above chelate composition, the second monomer unit is N,N-dimethyl-acrylamide.

[0077] In further embodiments of the above chelate composition, suitable addition-fragmentation chain transfer agents are independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

[0078] In a further embodiment of the above chelate composition, residues of the addition-fragmentation chain transfer agent are wholly or partially removed from the chelate composition after polymerization.

[0079] In further embodiments of the above chelating compositions, living animal cells prior to addition of the composition are affected by exposure to excess amounts of iron above normal in either or both of the extracellular or intracellular environments of the cells, where the excess iron contributes to chemical damage to the cells and disease in the animal, and the composition, when administered, partially reduces the excess iron levels that contribute to chemical damage to the cells, thereby reducing damage to the animal's cells.

[0080] In a further embodiment of the above chelate composition, the chelate composition has a molecular weight range defined by a low molecular weight limit as measured prior to iron binding of about 1500 daltons so as not to be normally incorporated into intracellular compartments within the cell membrane of living animal cells, and a high molecular weight limit that is sufficiently low to allow the composition to remain soluble in aqueous media.

[0081] In a further embodiment of the above chelate composition, the chelate composition comprises a metal binding chemical group of 3-hydroxy-pyridin-4-one incorporated into a support material consisting of pyrrolidone, acrylamide, imidazole or styrene.

[0082] In further embodiments of the above chelate compositions, the metal chelate compositions are for use in treating disease by administration to a cell(s), or to a human or other animal having a disease resulting from activity of a cell(s), and as a result of use of the composition, the external and internal aspects of the cell membrane and the internal aspects of the cell(s) underlying the cell membrane of the cell(s) are protected from chemically mediated damage caused in whole or in part by greater than normal amounts of iron in either the external environment or within the cell membrane of the cell(s).

[0083] In further embodiments of the above chelate compositions, the metal chelate compositions are for use in the treatment of one or more of the following human diseases: systemic lupus erythematosus-associated nephritis; rheumatoid arthritis; Parkinson's disease; Alzheimer's disease; Friedreich's ataxia; amyotrophic lateral sclerosis; Fanconi syndrome and related kidney diseases; type 2 diabetes mellitus; hemochromatosis; thalassemia; macular degeneration eye disease; a human cardiovascular disease or another autoimmune, metabolic, inflammatory or neurological disease, or a disease of another animal, the etiology of which is associated in part with higher than normal amounts of intracellular and / or extracellular iron contributing to the etiology of the disease.

[0084] In further embodiments of the above chelating compositions, the metal chelating composition is for administration in conjunction with a second iron chelator of molecular weight about <1500 Da, where the second iron chelator is normally taken up by a cell(s) and causes damage to the cell(s) associated with its properties of mobilizing and binding iron, and where the combined activity of the chelating composition and the second iron chelator reduces damage to the cell associated, at least in part, with excess reactive iron within the cell(s) or within the cell's external environment.

[0085] In one embodiment, the present disclosure provides a pharmaceutical composition comprising the above-described chelate composition and a pharma- ceutically acceptable carrier, excipient, or diluent, wherein the amount of the composition and the frequency and route of administration of the pharmaceutical composition are adjusted taking into consideration the particular disease being treated, such that the pharmaceutical composition addresses the relative amount of excess iron above normal that is addressed for the particular disease being treated.

[0086] In further embodiments of the above pharmaceutical compositions, the pharmaceutical composition contains a second iron chelator having a molecular weight of about <1500 Da selected from deferoxamine, deferasirox, deferiprone, SP-420, FBS701, or another iron chelator having a molecular weight of about <1500 Da, wherein the chelating composition reduces cell or tissue damage caused by the second chelator and improves the overall effectiveness of treating the disease.

[0087] In one embodiment, the present disclosure provides use of an iron chelating polymer for the treatment of a subject having a disease involving an iron-mediated pathology, or for the manufacture of a medicament for the treatment of a subject having a disease involving an iron-mediated pathology, The iron chelating polymer a reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent; The first monomer unit is compound (I):

[0088] [ka] is represented by At this time, R 1 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R 2 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; R 3 is independently selected from the group consisting of H and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12; the second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene; an iron chelating polymer is dissolved in an aqueous medium; the iron chelating polymer has a molecular weight of at least about 1500 Da prior to chelation; The iron chelating polymer contains one or more intramolecular hexadentate ligands for chelating iron.

[0089] In one embodiment, the present disclosure provides an iron chelating polymer for use in treating a subject having a disease involving an iron-mediated pathology, The iron chelating polymer a reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent; The first monomer unit is compound (I):

[0090] [ka] is represented by At this time, R 1 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 2 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 3 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12; the second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene; an iron chelating polymer is dissolved in an aqueous medium; the iron chelating polymer has a molecular weight of at least about 1500 Da prior to chelation; The iron chelating polymer contains one or more intramolecular hexadentate ligands for chelating iron.

[0091] In a further embodiment of the above use(s), or the iron chelating polymer(s) for the above uses, the one or more suitable metal binding chemical groups is a hydroxypyridinone:

[0092] [ka] In the formula, X, Y and Z are independently N or C, When X is N and Y and Z are C, When Y is N and X and Z are C, When Z is N, X and Y are C.

[0093] In further embodiments of the above use(s) or the iron chelating polymer(s) for the above uses, R 1 is H; R 2 is methyl; R 3 is methyl; n is 1 to 6, or optionally n is 2.

[0094] In a further embodiment of the above use(s) or the iron chelating polymer(s) for the above use, the first monomer unit is a compound (II):

[0095] [ka] It is represented by:

[0096] In a further embodiment of the above use(s), or the iron chelating polymer(s) for the above uses, the second monomer unit is 1-vinyl-2-pyrrolidone.

[0097] In a further embodiment of the above use(s), or the iron chelating polymer(s) for the above uses, the second monomer unit is N,N-dimethyl-acrylamide.

[0098] In further embodiments of the above use(s), or the iron chelating polymer(s) for the above uses, suitable addition-fragmentation chain transfer agents are independently selected from the group consisting of 2-ethoxythiocarbonylsulfanyl-propionic acid ethyl ester and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

[0099] In a further embodiment of the above use(s), or the iron-chelating polymer(s) for the above uses, residues of the addition-fragmentation chain transfer agent are wholly or partially removed from the iron-chelating polymer after polymerization.

[0100] In a further embodiment of the above use(s), or the iron chelating polymer(s) for the above uses, the iron chelating polymer reduces the extracellular concentration of free iron.

[0101] In further embodiments of the above use(s), or the iron chelating polymer(s) for the above uses, the iron chelating polymer reduces the intracellular concentration of free iron.

[0102] In further embodiments of the above use(s), or the iron chelating polymer(s) for the above uses, the disease is an autoimmune, metabolic, inflammatory or neurological disease.

[0103] In further embodiments of the above use(s), or the iron chelating polymer(s) for the above uses, the disease is one or more of systemic lupus erythematosus-associated nephritis, rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, Fredreich's ataxia, amyotrophic lateral sclerosis, Fanconi and related kidney diseases, nephritic kidney diseases observed in various autoimmune diseases such as lupus erythematosus and rheumatoid arthritis, diabetes mellitus type 2, hemochromatosis, thalassemia, macular degenerative eye disease, or cardiovascular disease.

[0104] In further embodiments of the above use(s), or the iron chelating polymer(s) for the above uses, the iron chelating polymer further comprises an iron chelating compound having a molecular weight of less than about 1500 Da.

[0105] In further embodiments of the above use(s), or the iron chelating polymer(s) for the above uses, the iron chelating compound is selected from deferoxamine, deferasirox, deferiprone, SP-420, FBS701, and MAHMP.

[0106] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the present disclosure. [Brief description of the drawings]

[0107] Embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. [Figure 1] Figure 1 shows UV-vis spectra showing MLCT bands for iron loading of DIBI. [Diagram 2] Relative changes in absorbance intensity at 460 nm for [Fe(MAHMP)3]DIBI = 790 μM (closed circles, concentration of Fe complex formed with the MAHMP moiety in DIBI), [Fe(MAHMP)3] = 690 μM (closed squares), and [Fe(DFP)3] = 637 μM (closed triangles) are shown after addition of DT to the corresponding control solutions in 0.1 M MOPS and pH 7.4. [Diagram 3] Fluorescence scans showing data from a representative experiment (top) and mean fluorescence intensity values ​​± standard error of the mean (SEM) from three independent experiments are shown and plotted as bar graphs (bottom). * indicates statistical significance using two-way ANOVA and Bonferroni post-hoc test. Data show that DIBI reduces intracellular labile reactive iron pools in macrophages. RAW 264.7 macrophages were loaded with 0.5 μM calcein-AM and seeded in 6-well plates at 250.000 cells / well in medium alone or medium containing the indicated concentrations of DIBI or PVP. After 4, 24 and 48 h of culture, cells were harvested and fluorescence intensity was measured by flow cytometry. [Figure 4]The mean relative %ROS+SEM data from three independent experiments are shown. Fig. 4A RAW 264.7 macrophages were stained with 10 μM CM-H2DCFDA and cultured for 24 h in medium alone or medium containing 200 μM DIBI or 1.28 mg / mL PVP in the absence or presence of 1 μg / mL LPS. Cell fluorescence was then measured by flow cytometry. Fig. 4B RAW 264.7 macrophages were cultured for 24 h in medium alone or medium containing 200 μM DIBI or 1.28 mg / mL PVP in the absence or presence of 100 ng / mL LPS and NO concentration in cell-free medium was determined. Statistical significance was determined by two-way ANOVA and Bonferroni post-hoc test. *p<0.05. Data show that DIBI reduces macrophage synthesis of ROS and NO species. [Diagram 5] The mean fold expression ± SEM for three independent experiments is shown. RAW 264.7 macrophages were cultured for 6 hours with medium alone or medium containing the indicated concentrations of DIBI or PVP and 100 ng / mL LPS. Cytokine mRNA expression relative to medium alone controls was determined by qRT-PCR. In the absence of LPS stimulation, little or no mRNA encoding cytokines was detected (data not shown). * indicates statistical significance using two-way ANOVA and Bonferroni post-hoc test. The data show that DIBI, but not PVP, suppresses RAW 264.7 macrophage expression of IL-1β, IL-6, and IFN-β mRNA, but not TNF-α mRNA. [Figure 6] Means ± SEM of four independent experiments are shown. RAW 264.7 macrophages not stimulated with LPS showed little or no cytokine production, which was not altered by the addition of DIBI or iron (data not shown). Statistical significance was determined by ANOVA with Tukey's multiple comparison post-hoc test. *p<0.05. The data show that exogenous iron reverses the inhibitory effect of DIBI on macrophage synthesis of IL-6. [Figure 7]Figure 1 shows UV-vis absorption of holo-transferrin ([holo-transferrin] = 47.5 µM) solutions during a DFP ([DFP] = 8.3 mM) and DIBI ([MAHMP]DIBI = 1.2 mM) dialysis experiment, demonstrating iron(III) uptake by DIBI during the initial dialysis step using DFP as an iron(III) shuttle between holo-transferrin and DFP. [Figure 8] Effect of DIBI on LPS-stimulated IL-6 secretion from CF15 cells. Apical (FIG. 8A) and basal (FIG. 8B). After polarization, CF15 cells were loaded with lipopolysaccharide (LPS; 200 ng / mL) and treated with DIBI (25 μM, 50 μM, 100 μM, 200 μM) or medium alone for 24 h. Supernatant IL-6 amounts (secreted) were normalized to total protein concentration and data shown represent the percentage change from control levels (medium only, dashed line) corresponding to mean values ​​of 526.5 pg / mL and 99.53 pg / mL of IL-6 in the apical and basal compartments, respectively. Data are presented as mean ± SD (N=3–4 independent experiments / group). *P<0.05 vs. LPS. LD25:LPS+DIBI 25M, LD50:LPS+DIBI 50M, LD100:LPS+DIBI 100M, LD200:LPS+DIBI 200M. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] One or more exemplary embodiments have been described by way of example. It will be understood that the embodiments and examples are provided for illustrative purposes intended for those of ordinary skill in the art and are not meant to be limiting in any way. All references to embodiments, examples, aspects, formulas, compounds, compositions, solutions, kits, and the like are intended to be illustrative and non-limiting.

[0109] A commonality to the other diverse diseases listed in Table 3 may be an excess of normal levels of labile reactive (free) iron. At least part of the pathology of these diseases with iron dysregulation may be due to incompletely coordinated iron, with complete iron coordination being essentially hexadentate. This excess iron, if not chaperoned, may be chemically unstable and reactive within the cell and in the immediate environment of the cells of the diseased tissue. In this regard, labile reactive iron may be a common feature underlying many other diverse diseases, and reactive iron above normal may contribute to the generation of otherwise safe levels of reactive oxygen species (ROS) above normal levels, which in turn may cause cell damage, tissue damage, and cell damage through chemical oxidation reactions due to oxidative stress. This damage from ROS may explain at least part of the pathology of these diseases. The diseases are referred to herein as diseases with iron-mediated pathology. A non-exhaustive list of diseases with iron-mediated pathology is also provided in Tables 2 and 3.

[0110] Iron requires hexadentate coordination to be completely chemically stable and is essentially chemically unreactive, for example for ROS generation. The use of chelating molecules that do not provide complete hexadentate coordination of iron, such as that bound by a single chelating agent molecule, may cause problems. With respect to deferoxamine, at least a portion of the iron bound by the deferoxamine starch conjugate composition may not be fully coordinated, may be chemically reactive, and may be chemically available for ROS formation. Given the nature of the starch carrier used, such deferoxamine starch conjugate composition may provide incompletely chaperoned iron, i.e., it may bind iron relatively weakly in reactive form without deferoxamine itself.

[0111] Some chelators known in the art, such as deferiprone, may require three molecules of chelator to fully coordinate to just one iron atom, and depending on the concentration of deferiprone applied and the iron concentration in the body compartment to which deferiprone reaches for treatment, partially coordinated iron is expected, and this incompletely coordinated iron may then chemically react and generate cell / tissue damaging ROS radicals, i.e., in or around cells and tissues exposed to the chelator. This ROS reactivity of incompletely deferiprone coordinated iron has been demonstrated to occur in vitro under conditions that may occur within the cells of the body (Devanur et al., 2008).

[0112] From Table 5, it can be seen that the current clinically used iron chelators, deferasirox and deferiprone, as well as the proposed SP-420 chelator, each require either two or three chelator molecules to fully fill only one iron atom for the tris bidentate coordination, i.e., to ensure that the bound iron atom is not available to participate in other chemical reactions that may, for example, generate cytotoxic ROS. This may at least partially explain the significant toxicity that has been reported for these various chelators, as summarized in Table 5.

[0113] Excessive, i.e., above normal levels, ROS generation within or around tissue cells may be at least partially related to the pathogenesis of a variety of non-infectious / non-cancer diseases, such as those listed by way of example in Table 3. These diseases have in common aspects of dysregulated iron metabolism, with some degree of greater than normal amounts of potentially chemically reactive iron, and this excess iron may result in excess ROS generation, leading to cell and tissue damage (i.e., damage as observed in the pathogenesis of the disease).

[0114] When humans or animals are treated with such ROS-promoting iron chelators, the use of iron chelators that are not fully coordinated and can bind iron in a ROS-reactive manner may partially generate ROS cell- and tissue-damaging species. Thus, when humans or other animals already experience iron-related ROS cell- and tissue damage as part of the pathogenesis of the disease, i.e., in the case of diseases associated with excess reactive iron above normal levels, treatment of these diseases with chelators that may themselves be incompletely bound to iron and in a ROS-promoting state may create further problems of ROS damage and toxicity. This problem severely limits the use of any iron chelator that does not substantially fully coordinate iron with / on a single chelator molecule, i.e., leaves iron substantially unavailable to promote ROS generation. The ideal chelator for treating these diseases is one that fully coordinates iron on or within a single molecule, for example, via one or more intramolecular ligands.

[0115] Physiologically normal amounts of iron-mediated ROS in cells of the human body may be important for normal cellular function, particularly as related to cells involved in defense against invading microorganisms. For example, defensive macrophages ingest invading bacteria and kill the invaders in a sac-like structure of the macrophage called a phagosome. This is in part due to the application of a normal, desired and useful amount of ROS as generated within the phagosome of the macrophage, which beneficially damages and kills the invading microorganism engulfed by the macrophage. The ROS generated within the phagosome is further spatially separated or compartmentalized by the phagosomal membrane from other intracellular aspects that may be subject to ROS damage. Thus, while it is undesirable to completely suppress ROS generation in defensive cells, such as macrophages, at the same time, excessive ROS generation (i.e., above normal requirements) may lead to both cell and tissue damage in the host, as seen in cells and tissues of the various diseases listed above. In this regard, macrophages may also play an important role in the diseases listed above. Macrophages play various important roles in both iron metabolism and its homeostatic regulation, as well as in host defense against invaders and in the cleansing and repair of damaged cells and tissues (Sukhbaatar and Weichhart, 2018).

[0116] Iron chelators for treating diseases with iron-mediated pathology are water-soluble chelators with a molecular size large enough to reach the extracellular environment in the immediate vicinity of cells of human tissues and animals with the disease to be treated, but not to be taken up into the internal aspects of the cells. Such polymers can sufficiently reduce the amount of reactive iron in the immediate environment of the cells, such as with macrophages, to suppress excess cell and tissue damaging ROS produced, while still ensuring the normal production of ROS in the appropriate amount required within the cells. In this regard, the reduction of the excess amount of extracellular reactive iron in the extracellular environment of the cells by iron chelating polymers over the normal amount can make less iron available for uptake by the cells to be treated. The excess intracellular amount of reactive iron taken up from the extracellular environment of the cells can then be reduced, resulting in a reduction in the excess amount of damaging ROS without substantially affecting the desired and necessary amount of ROS production, such as the ROS required for killing invading microorganisms in macrophages.

[0117] The nature and choice of the chelating polymer support material can be of substantial importance in order to avoid weakly bound, reactive iron that can promote ROS and cell damage. An ideal support material for a chelating polymer supports the addition of chelating groups without itself binding iron in a non-chaperoned form and without interfering with the iron binding of added chelating groups as immobilized or incorporated on or into the support material.

[0118] In various embodiments, iron chelating polymers are provided that are more easily capable of completely filling the complete iron chemical coordination, i.e., contain one or more hexadentate ligands, are water soluble, and are of a molecular size large enough so that they are not normally taken up by the cells of the body. These polymers can reduce excessive intracellular and / or extracellular reactive iron concentrations that would otherwise contribute to excessive, i.e., above normal, iron-associated ROS production. The reduction in excess ROS production (i.e., above normal amounts required) can then reduce cell and tissue damage caused by excess ROS for the disease being treated with the polymer.

[0119] The polymers described herein are useful for treating diseases in humans or other animals in which excess (i.e., greater than normal amounts of chemically reactive iron in the environment or within cells of humans or other animals) contributes to excess (i.e., greater than normal amounts of ROS caused by excess reactive iron), which excess ROS causes cell and tissue damage. Diseases for treatment include, but are not limited to, various autoimmune, neurological, metabolic, and inflammatory diseases.

[0120] Disclosed herein is an iron-chelating polymer for the treatment of diseases having iron-mediated pathologies. The polymer comprises the reaction product of a first monomer unit and a second monomer unit polymerized by a reversible addition-fragmentation chain transfer mechanism using a suitable addition-fragmentation chain transfer agent.

[0121] The first monomer unit is compound (I):

[0122] [ka] is represented by During the ceremony, R 1 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 2is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; R 3 is independently selected from the group consisting of H, alkyl, and alkyl optionally substituted with one or more of O, N, or S; n is 1 to 12; The second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethyl-acrylamide, ethyl methacrylate, N-vinylimidazole, and styrene.

[0123] The iron chelating polymer is water soluble. The iron chelating polymer has a molecular weight of at least about 1500 Da before chelation. The iron chelating polymer contains one or more intramolecular hexadentate ligands, for example, three ligands, for chelating iron. In such an embodiment, one polymer can bind three free iron ions.

[0124] In yet another embodiment, the polymer can be prepared by a reversible addition-fragmentation chain transfer polymerization mechanism using a suitable addition-fragmentation chain transfer agent, wherein the first monomer unit has the formula:

[0125] [ka] and one or more suitable hydroxypyridinone metal binding chemical groups, In the formula, X, Y and Z are independently N or C, When X is N and Y and Z are C, When Y is N and X and Z are C, When Z is N, X and Y are C.

[0126] In still further embodiments, the polymer may be prepared from a first monomer unit represented by compound (II) and a second monomer unit comprising either 1-vinyl-2-pyrrolidone or N,N-dimethyl-acrylamide.

[0127] [ka]

[0128] In further embodiments, the iron chelating polymers typically have a minimum molecular weight of about >1500 Da so as not to be incorporated into intracellular contexts within the cell membrane of living animal cells, and can bind iron up to full chemical coordination of the bound iron on or within a single molecule of the polymer. The iron chelating polymers may have an upper molecular weight limit small enough so as to remain soluble in aqueous solution with the bound iron.

[0129] In further embodiments, the metal iron chelating polymers are used to treat disease in a cell(s) or in a human or other animal having a disease caused by activity of a cell(s), where the polymer binds excess, above normal amounts of reactive iron, and as a result of using the polymer, external and internal aspects of the cell membrane as well as internal aspects of the cell underlying the cell membrane of a living cell are protected from chemically mediated damage caused in whole or in part by excess, above normal amounts of chemically reactive iron either in the external environment of the cell membrane of the cell(s) or within the cell membrane. In this regard, excess chemically reactive iron contributes to excess, i.e., excess, above normal amounts of ROS, which cause damage to cells or tissues as part of the disease in a human or another animal.

[0130] In further embodiments, the disease with an iron-mediated pathology treated by administration of an iron chelating polymer may be one or more of systemic lupus erythematosus, including associated nephritis; rheumatoid arthritis; Parkinson's disease; Alzheimer's disease; Friedreich's ataxia; amyotrophic lateral sclerosis; Fanconi syndrome and related kidney diseases; type 2 diabetes mellitus; hemochromatosis; thalassemia; macular degeneration eye disease; cardiovascular disease, or another autoimmune, metabolic, inflammatory or neurological disease, a disease of a human or other animal whose etiology is associated in part with higher than normal amounts of intracellular and / or extracellular iron contributing to the etiology of the disease.

[0131] In further preferred embodiments, the iron chelating polymer binds iron and remains substantially soluble with bound iron in the external cellular environment of a living animal cell, thereby reducing uptake of iron into intracellular aspects within the plasma membrane of the cell, such that the external and internal aspects of the plasma membrane of a living animal cell, as well as the intracellular aspects underlying the plasma membrane, are protected in part from chemically mediated damage such as that caused in whole or in part by excessive (i.e., above normal) amounts of iron and iron-associated reactive oxygen species (ROS) in either the external environment or within the plasma membrane of the animal cell.

[0132] In further aspects, the iron chelating polymer may be part of a pharmaceutical composition comprising the composition and a pharma- ceutically acceptable carrier, excipient, or diluent, and the amount of the pharmaceutical composition administered, as well as the frequency and route of administration of the pharmaceutical composition, are adjusted in view of the particular disease being treated, such that the pharmaceutical composition addresses the relative amount of excess iron above the normal amount to be addressed for the particular disease being treated.

[0133] In still further embodiments, the polymer does not block excessive normal amounts of necessary iron and iron-associated ROS activity (i.e., as required for normal cell function and bodily defenses); rather, the polymer reduces excess (i.e., above normal and damaging) amounts of iron, and consequently reduces excess (above normal) iron-associated ROS.

[0134] In yet further embodiments, the polymer is for use with or in combination with other known iron chelating compounds with molecular sizes of about <1500 Da. As discussed herein, such compounds may suffer from some degree of toxicity to animal cells or tissues, at least in part due to their molecular size and their binding of poorly coordinated iron, which may lead to iron-related toxic reactions. In this regard, the associated polymer may help partially overcome the toxicity limitations of other known chelating agents and provide improved overall iron-related therapeutic efficacy of the combination with reduced toxicity limitations. For example, deferoxamine, deferasirox, deferiprone, SP-420, FBS701, MAHMP, and other suitable compounds known in the art.

[0135] Patent Literature Holbein et al., U.S. Patent No. 10,709,784. Metal chelating compositions and methods for controlling the growth or activities of a living cell or organism.

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[0268] The patent and non-patent literature outlined herein is hereby incorporated by reference in its entirety. To the extent any incorporated subject matter conflicts with any disclosure herein, the disclosure herein shall take precedence over the incorporated material.

[0269] The present subject matter is further illustrated in the examples that follow. EXAMPLES

[0270] Example 1. Synthesis of a soluble copolymer chelating composition containing active pyridinone metal binding groups in the form of MAHMP copolymerized with vinylpyrrolidone using the RAFT procedure (US Pat. No. 11,059,785). A. Synthesis Procedure The composition of this example (Batch No. IS09865-025) illustrates optimized synthesis conditions in general with respect to obtaining high yield conversion of monomers to copolymer products, and with reference to the following synthesis scheme.

[0271] [ka]

[0272] In a 50 mL two-neck round-bottom flask equipped with a magnetic stirrer and a reflux condenser, dissolve N-vinyl-2-pyrrolidone (B) (3.5 g, 0.0315 mol) and MAHMP (A) (0.25 g, 0.00106 mol) in 4 mL of deionized water (E) and immerse the mixture in N 2 To this mixture was added RAFT agent (0.136 g, 0.000652 mol), 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid (ETSPA) (F) and TMEDA (0.227 g, 0.0.00195 mol) (promoter (D)), and stirred under N 2 The entire mixture was degassed by purging with N for 20 minutes. After degassing, tert-butyl hydroperoxide (0.35 g, 0.00388 mol) (initiator (C)) was added to the mixture. The mixture was then heated to 40° C. and the reaction was continued at the same temperature for 18 hours under N 2 The reaction mass was placed in a rotary evaporator and water was removed under reduced pressure after 18 hours. The crude mass was dissolved in methanol and filtered to remove insoluble impurities and the filtrate was concentrated to about 50 wt% polymer solution. This solution was then slowly precipitated with 50 volume excess of MTBE under constant stirring. The precipitate was then filtered and dried on a rotary evaporator at 50°C under 35-50 mmHg for 4-6 hours until a constant weight of product copolymer (Batch ISO9865-025) was observed. The mass yield of MAHMP-pyrrolidone copolymer was 80% with respect to the mass of monomers (A+B) initially fed to the polymerization reaction and the copolymer was 1 It was found to contain a MAHMP content of 5.7% as determined by H NMR spectroscopy. The metal chelate copolymer was found to have a molecular weight (Mw) of about 7.3 kDa with a PDI (Mw / Mn) of 1.7 using GPC analysis.

[0273] B: Optimization of MAHMP. NVP monomer ratio With reference to the copolymer synthesis scheme and Example 1A above, a series of test polymerizations were conducted using the procedure of Example A (above) except that the total monomer (A+B) content was maintained at 50 equivalents for the polymerization reaction and the ratio of the metal chelating monomer MAHMP (A) to the comonomer NVP (B) was varied while the other chemical components (C), (D), (E) and (F) were kept the same for each test. The results in Table 6 below show that as the amount of MAHMP (A) fed to the polymerization increases relative to NVP (B), the overall copolymer yield decreases but the percentage of MAHMP content in the product copolymer increases. Ideally, a copolymer MAHMP content of 10-20% may be preferred for a chelating copolymer composition with high metal binding capacity, but maintaining high copolymer yield is also very important for efficiency of monomer usage. RAFT-mediated polymerizations using RAFT agent 4, 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid (ETSPA), were found to provide fairly narrow molecular weight distributions of the copolymer products, with the average Mw of the compositions being somewhat similar for the different MAHMP:NVP conditions. Based on these results, it appeared that MAHMP:NVP ratios between 1:10 and 1:15 provided the best overall MAHMP content (10-15%), with a polydispersity index (PDI) of about 1.4-2.0, while retaining a product yield of about 80%. The composition prepared by this synthetic procedure is referred to as DIBI for ease of reference with other examples provided.

[0274] [Table 6]

[0275] From these test syntheses, sample batch ISO9865-044 provided a relatively high copolymer yield (about 80%), allowing for about 15 mol % incorporation of MAHMP into the copolymer composition, and provided a relative (i.e., relative to a linear polystyrene calibration standard using gel exclusion chromatography) product Mw of 3600 Da. Separate true Mw determination of this composition (designated DIBI) by laser techniques indicated an actual true Mw of about 9 kDa.

[0276] Example 2. Demonstration of iron binding capacity and iron formation constants of the composition referred to in Example 1B as DIBI (Ang et al., 2018 and Gumbau-Brisa et al., 2020). A. Spectrophotometric characterization of Fe chelation by DIBI The dry DIBI composition prepared in Example 1B (30.3 mg, 3.4 μmol) was dissolved in 25 mL of MOPS buffer. A 1 mL aliquot of this solution was placed in a vial and diluted with 8.6 mM Fe(NO 3 ) 3 9H 2 Aqueous O solution was added to a series of vials. A corresponding amount of MOPS buffer was then added to each vial to reach a final reaction volume of 1.1 mL for each vial.

[0277] The solutions were gently shaken in a shaker plate for 24 h, at which point the absorbance of 300 µL of each vial was recorded from 350 to 800 nm. The results are shown in Figure 1.

[0278] A plot of the absorption maximum at 460 nm versus iron(III) concentration gave a plateau at 371 μM iron(III). As a result, the nine MAHMP residues present on average on each molecule of DIBI, i.e., H 1The results, measured separately by nuclear magnetic flux spectroscopy (Ang et al., 2018), showed that iron tris-chelation (i.e., hexadentate binding of three iron atoms per molecule of DIBI with nine bidentate MAHMP residues, three MAHMP moieties for each bound iron atom) was all available, resulting in a total iron-binding capacity of 338 μmol iron per gram of DIBI. No precipitate was observed during the iron titration experiments, and the colored film obtained upon solution evaporation readily redissolved in water after drying for several weeks. These results indicate that both DIBI and iron-loaded DIBI are soluble in aqueous media.

[0279] B. Iron(III) Binding Affinities of DIBI and MAHMP To determine the binding affinity of DIBI to iron(III) and its component chelating ligand MAHMP, acidic solutions of the corresponding 1:3 Fe:HPO complexes in solutions of constant ionic strength (I = 0.1 M) were titrated with NaOH. Titrations were performed under a humid nitrogen atmosphere to avoid concentration changes due to evaporation. Aliquots of each complex solution were taken periodically during the titrations, and UV-vis spectra of the aliquots were collected as a batch once the titration was complete. The pH range tested spanned the equilibrium between bis- and tris-coordination for the ligand, as these are the only two species expected at physiological pH. Fe 3 For -DIBI, spectra were collected 24 hours after the batch of aliquots was collected. Spectra were processed using HypSpec2014 to determine the iron(III) binding affinities of MAHMP, DIBI, and deferiprone (DFP) as a reference, as summarized in Table 7.

[0280] [Table 7] * The values ​​in brackets are the standard deviations of the last digit.

[0281] MAHMP appeared to be similar to DFP, as both are bidentate chelators, as might be expected given the close structural similarity; i.e., three chelator molecules are required for tris-bidentate coordination of one Fe(III). DIBI, on the other hand, has multiple MAHMP chelating monomers (an average of nine per molecule) dispersed along its non-iron-binding polymer backbone. Thus, formation of intramolecular tris complexes could result from chelating MAHMP monomers in spatial proximity along the polymer backbone backbone or from structural loops that bring MAHMP residues in sufficient proximity to one another. The enhanced iron binding of DIBI is due to a higher Logβ 2 and especially higher Logβ 3 As reflected in the formation constants, DIBI exhibits up to 1000-fold higher Fe-binding affinity than either MAHMP or DFP.

[0282] Example 3: Resistance of iron bound to the composition of Example 1B (DIBI) to chemical reduction by dithionite compared to the reducibility of deferiprone-bound iron (Gumbau-Brisa et al., 2020). The chemical reduction of iron from Fe(III) to Fe(II) is part of the iron chemical reduction cycle, which drives ROS generation in and around cells and also facilitates the removal of iron from intracellular stores for use elsewhere in the body. Generally, iron in the extracellular compartment of the body is in the Fe(III) form, which helps to limit iron-related toxic reactions, as free Fe(II) can be rapidly oxidized to Fe(III) and drive ROS generation, for example. Thus, the relative ability of an iron chelator to retain Fe(III) and resist reduction to Fe(II) would be of benefit. Correspondingly, the relative chemical reducibility of Fe(III) bound by deferiprone, MAHMP, and DIBI to Fe(II) was tested. Different iron-loaded chelators were first prepared such that just enough iron was used to ensure the initial presence of fully coordinated iron(III), i.e., a ratio of three chelator molecules of either deferiprone or MAHMP for each iron(III) atom present. DIBI in which MAHMP content was measured was similarly loaded with just enough iron to fill its MAHMP iron binding sites on a similar basis. The iron chelate was then added to the iron chemical reducer dithionite (DT, [S 2 O 4 ] 2- ) and the relative change in absorbance intensity at 460 nm was used to monitor iron reduction, the results of which are shown in FIG. 2.

[0283] Dithionite (DT) is Fe(DFP) 3 The DFP-bound iron(III) was readily reduced, as indicated by a decrease in the intensity of the band at 460 nm in the complex solution (Figure 2). The same reaction was observed for the trisFe(III) complex of MAHMP, and spectrophotometric characterization revealed Fe(DFP) 3 and Fe(MAHMP) 3 However, under the same conditions, the oxidation rates of Fe 3The DIBI complex was much more resilient to reduction of bound iron by DT, with only a slight loss of intensity of the 460 nm band observed. It is important to note that the spectral change was a decrease in the intensity of the 460 nm band, indicating iron(II) release from the complex, rather than a wavelength shift of the band maximum, which would indicate iron dissociation due to pH change. Thus, the solution showed a decrease in absorbance, rather than a color shift from red (the color of the Tris complex) to a different color. Given the similarity between the (HPO) iron binding sites for the three chelators tested, any differences in the behavior of iron-containing DIBI could be attributed to its tertiary structure, and the way it retains its bound iron. The results indicate that DIBI binds iron more tightly and in a more stable manner, and that the bound iron is not readily reduced to Fe(II). Based on these results, it is believed that the iron bound by DIBI under physiological conditions, such as in and around animal cells, is highly stable and therefore less available to promote ROS formation from the Fe(III) / Fe(II) reduction cycle activity of its bound iron.

[0284] Example 4. Demonstration of reduction of intracellular macrophage iron concentration by DIBI, a composition of Example 1B (Ghassemi-Rad, et al. 2022). The effect of DIBI on iron availability and intracellular concentration in macrophages was determined by comparing the intracellular pool of labile, i.e., chemically reactive, iron in RAW 264.7 macrophages cultured in the absence or presence of 200 μM DIBI. Another preliminary test by staining with the cell viability dye 7-AAD showed that 200 μM DIBI was not cytotoxic to RAW 264.7 mouse macrophages or freshly obtained mouse bone marrow-derived macrophages (BMDMs). RAW 264.7 macrophages were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine, 100 μg / mL streptomycin, 100 U / mL penicillin, and 5 mM HEPES buffer (pH 7.4), referred to as complete medium, at 37 °C in a humidified 5% CO2-incubated 10-well plate. 2The cells were maintained in an incubator at 37°C. The cells were passaged using a 25cm cell scraper. Macrophages were then plated in 6-well plates at 2.5 x 10 5 Cells were seeded at 100x the cell / well and cultured overnight to allow cell attachment. Cells were then treated with 0.5 μM calcein-AM and cultured at 37° C. for 30 min. Cells were then washed with room temperature PBS and treated with complete medium alone or complete medium containing 200 μM DIBI or 1.28 mg / mL polyvinylpyrrolidone (PVP0, i.e., as a control for the polymer backbone of DIBI). Cells that were not exposed to calcein-AM served as an additional control. After 4, 24, and 48 h of culture, cells were harvested and analyzed using the FL1 channel of a BD FACScanto™ flow cytometer (1×10 4 number of cells / sample). The fluorescence of calcein-AM entering the cells is quenched by intracellular free labile reactive iron such that lower iron levels correspond to higher measured fluorescence. As can be seen in Figure 3, RAW 264.7 macrophages cultured in the presence of DIBI showed a significant reduction in the intracellular pool of labile iron due to iron chelation by DIBI in the culture medium as determined by flow cytometric analysis of calcein-AM labeled cells. The effect was specific to the iron chelating activity of DIBI, since an equivalent concentration of PVP with a similar molecular weight, i.e., structural backbone component of the DIBI copolymer, did not reduce the intracellular pool of labile iron in RAW 264.7 macrophages.

[0285] This example demonstrates that the polymers disclosed herein can effectively reduce intracellular iron levels by binding iron in the extracellular environment immediately surrounding the cells being treated.

[0286] Example 5. Reduction of intracellular ROS and extracellular NO production in macrophages by DIBI, a composition of Example 1B (Ghassemi-Rad, et al. 2022). 2.5 x 10 RAW 264.7 cells 5Cells / well were seeded in 6-well plates and cultured overnight. The next day, cells were cultured in complete medium alone or complete medium containing 200 μM DIBI or 1.28 mg / mL PVP in the absence or presence of bacterial lipopolysaccharide (LPS) at 1 μg / mL. Under these conditions, macrophage ROS production was induced by the addition of LPS, a potent inflammatory agent. After 24 h of culture, cells were washed with PBS, resuspended in FBS and phenol red-free medium containing 10 μM CM-H2DCFDA, and incubated at 37°C in the dark for 30 min to detect intracellular ROS. Cells were then harvested with TrypLE™ Express and washed with room temperature PBS. Cells (1×104 cells / sample) were analyzed via the FL1 channel on a BD FACSCalibur™ flow cytometer (BD Biosciences, Mississauga, ON). Data were processed using FCS Express software (version 3.0, De Novo Software, Thornhill, ON).

[0287] For separate experiments to measure NO released by macrophages, production was stimulated using 100 ng / ml LPS, and at the end of the incubation, NO concentrations in cell-free culture supernatants were measured using a colorimetric Griess assay.

[0288] As shown in Figure 4, LPS-stimulated RAW 264.7 macrophages exhibited a four-fold increase in ROS compared to unstimulated control cells. In this experiment, 1 μg / mL LPS was used to induce vigorous production of ROS. Treatment with 200 μM DIBI reduced ROS production by LPS-stimulated RAW 264.7 macrophages, but not a similar concentration of PVP.

[0289] ROS, which are important for host defense and contribute to inflammatory diseases, are produced through the electron transport chain of mitochondria, cytochrome P450, and NADPH oxidase. The iron-dependent Fenton reaction is important for ROS generation, including the production of highly toxic hydroxyl radicals, and therefore intracellular ROS was reduced in LPS-stimulated macrophages treated with DIBI. NO is also normally produced by cells, but when in excess, can contribute to damage to cells. Importantly, DIBI did not completely inhibit ROS or NO production, but rather attenuated both excess ROS and NO production. This is significant considering that macrophage ROS- and NO-related killing of phagocytosed bacterial pathogens is also important in host defense. This example, in conjunction with the results of Example 4, shows that the polymers of the present disclosure reduce excess intracellular ROS production and overall NO production by lowering iron levels and reducing the excess production induced by the potent inflammatory LPS agent.

[0290] Example 6. Demonstration of reduction in excessive inflammatory cytokine production by macrophages using DIBI, a polymer of Example 1B (Ghassemi-Rad, et al. 2022). Macrophages prepared as in Example 6 were cultured for 6 hours in medium alone or medium containing the indicated concentrations of DIBI or PVP+100 ng / mL LPS to stimulate cytokine expression, after which cell macrophage mRNA expression for the main inflammatory cytokines IL-1β, IL-6, IFN-β, and TNF-α was examined, and the results are shown in FIG. 5.

[0291] As shown in Example 6, the reduction in ROS production by LPS-stimulated macrophages in the presence of DIBI may contribute to the reduction in IL-1β, IL-6 and IFN-β synthesis, since mitochondrial ROS promote LPS-induced pro-inflammatory cytokine synthesis. These various results are consistent with DIBI suppressing but not completely inhibiting an excessive LPS-induced inflammatory response, i.e., excessive ROS and cytokine production. These results further indicate that the PVP carrier material of the DIBI polymer had no effect.

[0292] Example 7. Iron-dependence of reduction of excessive inflammatory cytokine production by macrophages using the polymer DIBI of Example 1 (Ghassemi-Rad, et al. 2022). Macrophages were cultured in the absence or presence of 100 ng / mL LPS for 24 hours using medium alone or medium containing 200 μM DIBI and / or iron (Fe) as 1000 μM ferric citrate as in Example 6. Cell-free supernatants were collected and IL-6 was measured by ELISA, and the results are shown in FIG.

[0293] These results indicate that the inhibition of excessive IL-6 cytokine production by DIBI was directly related to its iron chelating activity in the immediate environment of macrophages, since the addition of excess iron to fully saturate the added DIBI iron-binding capacity reversed the inhibitory effect of DIBI.

[0294] Example 8. Removal of deferiprone-bound iron and binding of iron from deferiprone to DIBI (polymer of Example 1B) (Gumbau-Brisa et al, 2020). DIBI to Fe(DFP) 3 to a solution of 1.0 kDa DIBI such that both chelators were equimolar in terms of hydroxypyridinone moiety content, and the resulting solution was stirred at room temperature for 24 hours. The reaction solution was dialyzed to separate DIBI from deferiprone based on their different molecular weights. This was achieved by using a dialysis tubing bag with a pore cutoff of 3.5 kDa MW, allowing deferiprone to pass through the membrane but preventing the passage of DIBI. After a further 24 hours of incubation, a colored dialysis bag retentate solution (DIBI solution) and a colorless dialysate DFP solution were obtained. Starting Fe(DFP) 3 Comparison of the UV-vis spectra of the -DIBI solution and the DIBI dialysis retentate showed that the HPO content (band at 280 nm) was reduced by approximately half, while the iron complex content (broad band at 460 nm) remained constant. 2 -3DIBI is retained by the dialysis membrane and the complex Fe(DFP)3 was readily dialysable. This result indicates that DIBI can displace iron(III) from the coordination environment of DFP and binds the displaced iron from DFP due to the higher iron-binding strength of DIBI.

[0295] Example 9: Removal of iron bound to transferrin and its conjugation to DIBI, a polymer of Example 1B (similar to Gumbau-Brisa et al., 2020). Transferrin has biological relevance as the major extracellular vertebrate host iron-binding, shuttle and iron chaperone protein, acting during infection, inflammation and diseases resulting from iron dysregulation as disclosed in this application. A solution of DIBI in a dialysis tubing bag was dialyzed against a solution of holo-transferrin (iron-loaded form of transferrin) and DFP in MOPS / bicarbonate buffer at pH 7.4. The dialysis membrane has an exclusion limit of 3.5 kDa, ensuring that transferrin (MW 80 kDa) and DIBI (MW 9 kDa) remained separated on opposite sides of the membrane, while DFP, being a small molecule (MW 139 Da), moved freely back and forth through the membrane. After reacting the mixture for 48 hours at 8°C, the DIBI solution in the dialysis tubing bag was orange in color and the holo-transferrin DFP solution outside the dialysis bag was pale orange in color. The DIBI solution was then dialyzed for another 24 hours in MOPS / bicarbonate buffer at pH 7.4, after which the UV-vis spectrum of the solution was recorded as shown in FIG. 7. A band at 460 nm in the first UV-vis spectrum of the holo-transferrin solution indicated that transferrin was bound to iron(III) ions. At the end of the experiment, the intensity of the band decreased, indicating loss of iron(III) from transferrin. After the second dialysis, the DIBI solution showed a band at 460 nm, as expected for coordination of iron(III) ions to DIBI.

[0296] This example further shows that DIBI has a higher affinity for iron than transferrin, and on this basis, DIBI can be expected to enhance natural iron binding and iron chaperoning by transferrin, as required to prevent inappropriate production of ROS, i.e., when iron is present at higher than normal levels, such as in the diseases disclosed herein.

[0297] This example further demonstrates the utility of using a low molecular weight chelator such as deferiprone in conjunction with the disclosed polymers to act as an iron shuttle to more rapidly load the polymer with iron. DIBI, which removes iron from deferiprone, reduces the possible formation of reactive iron-deferiprone species that may be involved in ROS formation due to the incompletely coordinated, and therefore reactive, iron available on the iron-deferiprone species.

[0298] Example 10. Freedom of toxicity of the DIBI polymer of Example 1B. The systemic toxicity of DIBI, the polymer prepared in Example 1B, was evaluated by daily administration via intravenous route to both male and female rats for 14 consecutive days.

[0299] The dose administered was 5 mL / kg body weight. Each toxicity study group consisted of 5 rats / sex / group. No pre-terminal mortality or treatment-related clinical signs were observed in either male or female rats treated with DIBI at 50 mg / kg / day, 100 mg / kg / day, or 200 mg / kg / day. No treatment-related effects were observed in the mean body weight of male or female rats administered DIBI at 50 mg / kg / day, 100 mg / kg / day, or 200 mg / kg / day. No changes in food intake were observed in animals treated with DIBI at 50 mg / kg / day, 100 mg / kg / day, or 200 mg / kg / day.

[0300] Evaluation of clinical chemistry parameters showed no significant changes in treated rats compared to untreated control rats. There were no changes in hematological parameters including total red blood cell count, total white blood cell count, hematocrit, and hemoglobin relative to control rats.

[0301] No gross or histopathological changes were observed in either male or female rats treated with DIBI at 50 mg / kg / day, 100 mg / kg / day, and 200 mg / kg / day, based on examination of the adrenal glands; brain (cerebrum, cerebellum, pons); cecum; colon; duodenum; femur; heart; ileum; jejunum; kidneys; liver; lungs; mesenteric lymph nodes; rectum; spleen; stomach; sternum; thymus, and testes / ovaries.

[0302] Based on the results, and in the absence of life-threatening pathological observations, the no observed adverse effect level (NOAEL) for DIBI was considered to be greater than 200 mg / kg / day (i.e., the highest dose tested in this study).

[0303] Example 11. The polymer of Example 1B (DIBI) suppresses excessive cytokine production in airway epithelial cells from subjects with cystic fibrosis (CF) (Aali et al., 2020).

[0304] A CF nasal epithelial cell line (JME / CF15), homozygous for the ΔF508 mutation in the CFTR gene, was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.088% transferrin, 0.088% T3 hormone (triiodothyronine), 0.0088% EGF (epidermal growth factor); 1.76% hydrocortisone; 0.044% insulin; 0.088% epinephrine; and 0.176% adenine. Cells were grown in T25 tissue culture flasks at 37° C., 5% CO. 2Cells were grown in a 5% CO atmosphere until confluence was reached. Once confluent, cell cultures were detached with 0.1% trypsin-EDTA and seeded in transwells (24 mm diameter, 0.4 μm diameter pores) with both apical and basolateral compartments filled with 2 mL of the described culture medium. After 1 week, a dense cell monolayer was formed, after which the apical medium was removed. The monolayer was then maintained under air-liquid interface (ALI) conditions to achieve polarization and differentiation. After removing the apical medium, the medium in the basolateral compartment was replaced every 2 days until the cells reached a dry apical surface and were considered ready for stimulation. Following cell polarization and tight junction (TJ) formation, transwells were designated as control (medium) or for stimulation with the bacterial toxin lipopolysaccharide (LPS) with or without co-treatment with DIBI (LPS+DIBI) or DIBI alone (DIBI). Stimulation with LPS (from Escherichia coli, serotype O26:B6; Sigma-Aldrich, Oakville, ON) was performed in fresh phenol red-free medium at concentrations of 100 ng / mL, 200 ng / mL, or 300 ng / mL for bidirectional stimulation of cells. Once optimal LPS stimulation conditions were established (24 h dose), 200 ng / mL LPS exposure was found to be optimal. The following experimental groups were tested: control (medium only); LPS (200 ng / mL); LPS+DIBI (25 μM, 50 μM, 100 μM, or 200 μM) and DIBI only (25 μM, 50 μM, 100 μM, or 200 μM). The appropriate stimulation medium was applied to both the apical and basolateral surfaces in a volume of 0.5 mL and 2.5 mL, respectively. Cells were incubated at 37 °C and 5% CO 2The cells were incubated in a 5% CO2-free environment for 24 hours before harvesting. IL-6 levels in apical and basolateral cell culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits. A monoclonal human IL-6 antibody (Invitrogen, Carlsbad, CA) was used. Cytokine levels were normalized to the total protein concentration of lysed cells measured by Bradford Colorimetric Assay. Figure 8 shows the stimulation of IL-6 cytokine production and release caused by LPS, and the inhibition of excess IL-6 release by DIBI from both the apical (A) and basal (B) cell surfaces.

[0305] IL-6 is a key cytokine associated with pro-inflammatory responses and increases ROS production as shown for macrophages in Examples 5 and 6. For CF airway cells, it is known that ROS damage mediated by excess, i.e., above normal, reactive iron is part of the pathogenesis of CF disease, i.e., associated with ROS-mediated cell and tissue damage in the airways of CF subjects. This example shows that DIBI, which absorbs excess above normal amounts of reactive iron, dose-dependently suppresses the excessive inflammatory response induced by LPS in association with cytokine production. In this context, there is a similar attenuation in ROS activity as demonstrated for the iron-specific attenuation by DIBI of excess IL-6 and ROS activity in macrophages as shown in Figures 4, 5, and 6. LPS is a potent inflammatory agent released from the cell walls of gram-negative bacteria such as Pseudomonas aeruginosa, which have been shown to typically colonize the airways of CF subjects.

[0306] What has been described is merely illustrative of the application of the principles of the present disclosure, however, it will be apparent to one skilled in the art that certain variations and modifications can be made without departing from the scope of the claims set forth below.

Claims

1. The use of iron chelate polymers for the treatment of subjects with diseases involving iron-mediated pathologies, The aforementioned iron chelate polymer The reaction product comprises a first monomer unit and a second monomer unit polymerized by a reversible addition-cleavage chain transfer mechanism using a suitable addition-cleavage chain transfer agent, The first tourist unit is compound (I): 【Chemistry 1】 Represented by, During the ceremony, R １ This is independently selected from the group consisting of H and alkyl groups optionally substituted with one or more of O, N, or S. R ２ This is independently selected from the group consisting of H and alkyl groups optionally substituted with one or more of O, N, or S. R ３ This is independently selected from the group consisting of H and alkyl groups optionally substituted with one or more of O, N, or S. n is between 1 and 12. The second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethylacrylamide, ethyl methacrylate, N-vinylimidazole, and styrene. The iron chelate polymer is dissolved in an aqueous medium. The iron chelate polymer has a molecular weight of at least about 1500 Da before chelation. The iron chelate polymer is used to include one or more intramolecular hexadentate ligands for chelating iron.

2. The use of iron chelate polymers in the manufacture of pharmaceuticals for the treatment of subjects with iron-mediated diseases, The aforementioned iron chelate polymer The reaction product comprises a first monomer unit and a second monomer unit polymerized by a reversible addition-cleavage chain transfer mechanism using a suitable addition-cleavage chain transfer agent, The first tourist unit is compound (I): 【Chemistry 2】 Represented by, During the ceremony, R １ This is independently selected from the group consisting of H and alkyl groups optionally substituted with one or more of O, N, or S. R ２ This is independently selected from the group consisting of H and alkyl groups optionally substituted with one or more of O, N, or S. R ３ This is independently selected from the group consisting of H and alkyl groups optionally substituted with one or more of O, N, or S. n is between 1 and 12. The second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethylacrylamide, ethyl methacrylate, N-vinylimidazole, and styrene. The iron chelate polymer is dissolved in an aqueous medium. The iron chelate polymer has a molecular weight of at least about 1500 Da before chelation. The iron chelate polymer is used to include one or more intramolecular hexadentate ligands for chelating iron.

3. The one or more suitable metallic chemical groups mentioned above are hydroxypyridinone: 【Transformation 3】 And, In the formula, X, Y, and Z are independently N or C if, If X is N and Y and Z are C, If Y is N, and X and Z are C, The use according to claim 1, wherein Z is N and X and Y are C.

4. R １ H is, R ２ It is methyl, R ３ is methyl, The use according to claim 1, wherein n is 1 to 6, and n is optionally 2.

5. The first monomer unit is compound (II): 【Chemistry 4】 The use described in claim 1, as represented by...

6. The use according to claim 1, wherein the second monomer unit is 1-vinyl-2-pyrrolidone.

7. The use according to claim 1, wherein the second monomer unit is N,N-dimethylacrylamide.

8. The use according to claim 1, wherein the appropriate addition-cleavage chain transfer agent is independently selected from the group consisting of ethyl 2-ethoxythiocarbonylsulfanyl-propionate and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

9. The use according to claim 1, wherein the residue of the addition-cleavage chain transfer agent is removed entirely or partially from the iron chelate polymer after polymerization.

10. The use according to claim 1, wherein the iron chelate polymer reduces the extracellular concentration of free iron.

11. The use according to claim 10, wherein the iron chelate polymer reduces the intracellular concentration of free iron.

12. The use according to claim 1, wherein the disease is an autoimmune disease, a metabolic disease, an inflammatory disease, or a neurological disease.

13. The use according to claim 12, wherein the disease is one or more of the following: systemic lupus erythematosus-associated nephritis, rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, Fredreich's ataxia, amyotrophic lateral sclerosis, Fanconi and related kidney disease, type 2 diabetes mellitus, hemochromatosis, thalassemia, macular degenerative eye disease, or cardiovascular disease.

14. The use according to any one of claims 1 to 13, further comprising an iron chelate compound having a molecular weight of less than approximately 1500 Da.

15. The use according to claim 14, wherein the iron chelate compound is selected from deferoxamine, deferasirox, deferipron, SP-420, FBS701, and MAHMP.

16. An iron chelate polymer for use in the treatment of subjects with diseases involving iron-mediated pathologies, The aforementioned iron chelate polymer The reaction product comprises a first monomer unit and a second monomer unit polymerized by a reversible addition-cleavage chain transfer mechanism using a suitable addition-cleavage chain transfer agent, The first tourist unit is compound (I): 【Transformation 5】 Represented by, During the ceremony, R １ This is independently selected from the group consisting of H, alkyl, and alkyls optionally substituted with one or more of O, N, or S. R ２ This is independently selected from the group consisting of H, alkyl, and alkyls optionally substituted with one or more of O, N, or S. R ３ This is independently selected from the group consisting of H, alkyl, and alkyl substituted with one or more of O, N, or S. n is between 1 and 12. The second monomer unit is independently selected from the group consisting of 1-vinyl-2-pyrrolidone, acrylic acid, methyl methacrylate, N,N-dimethylacrylamide, ethyl methacrylate, N-vinylimidazole, and styrene. The iron chelate polymer is dissolved in an aqueous medium. The iron chelate polymer has a molecular weight of at least about 1500 Da before chelation. The iron chelate polymer is an iron chelate polymer comprising one or more intramolecular hexadentate ligands for chelating iron.

17. The one or more suitable metallic chemical groups mentioned above are hydroxypyridinone: 【Transformation 6】 And, In the formula, X, Y, and Z are independently N or C if, If X is N and Y and Z are C, If Y is N, and X and Z are C, The iron chelate polymer for use according to claim 16, wherein Z is N and X and Y are C.

18. R １ H is, R ２ It is methyl, R ３ It is methyl, An iron chelate polymer for use according to claim 16, wherein n is 1 to 6.

19. An iron chelate polymer for use according to claim 18, wherein n is 2.

20. The first monomer unit is compound (II): 【Transformation 7】 An iron chelate polymer for use according to claim 16, as represented by [the specified figure].

21. The iron chelate polymer for use according to claim 16, wherein the second monomer unit is 1-vinyl-2-pyrrolidone.

22. The iron chelate polymer for use according to claim 16, wherein the second monomer unit is N,N-dimethylacrylamide.

23. The iron chelate polymer for use according to claim 16, wherein the appropriate addition-cleavage chain transfer agent is independently selected from the group consisting of ethyl 2-ethoxythiocarbonylsulfanyl-propionate and 2-ethoxythiocarbonylsulfanyl-2-methyl-propionic acid.

24. The iron chelate polymer for use according to claim 16, wherein residues of the addition-cleavage chain transfer agent are removed whole or partially from the iron chelate polymer after polymerization.

25. The iron chelate polymer for use according to claim 16, wherein the iron chelate polymer reduces the extracellular concentration of free iron.

26. The iron chelate polymer for use according to claim 25, wherein the iron chelate polymer reduces the intracellular concentration of free iron.

27. The iron chelate polymer for use according to claim 16, wherein the disease is an autoimmune disease, a metabolic disease, an inflammatory disease, or a neurological disease.

28. The iron chelate polymer for use according to claim 27, wherein the disease is one or more of the following: systemic lupus erythematosus-associated nephritis, rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, Fredreich's ataxia, amyotrophic lateral sclerosis, Fanconi and related kidney disease, type 2 diabetes mellitus, hemochromatosis, thalassemia, macular degenerative eye disease, or cardiovascular disease.

29. An iron chelate polymer for use according to any one of claims 16 to 28, further comprising an iron chelate compound having a molecular weight of less than approximately 1500 Da.

30. The iron chelate polymer for use according to claim 29, wherein the iron chelate compound is deferoxamine, deferasirox, deferipron, SP-420, FBS701, or MAHMP.

31. A method for treating a disease involving an iron-mediated pathology in a subject, comprising the step of administering the iron chelate polymer described in any one of claims 16 to 24 to the subject.

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