Treatment of cerebral copper disorders

Abstract

The present disclosure relates to the treatment and prevention of cognitive impairment, cognitive decline and / or dementia or symptoms of brain neurodegeneration in subjects with diabetes by administering a pharmaceutical composition comprising a compound capable of lowering the level or amount of copper in the hippocampus or elsewhere in the brain of the subject.The present invention provides, for example, a method for treating cognitive impairment in a subject with type 2 diabetes, comprising administering to the subject a pharmaceutical composition comprising a compound capable of reducing copper in the subject, wherein urinary copper is increased after administration of the composition, and cognitive impairment in the subject is alleviated.

Description

[Technical field]

[0001] STATEMENT OF RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 332,551, filed April 19, 2022, the entire contents of which are incorporated by reference herein for all purposes. Field The present invention relates to diabetes, dementia, cognitive impairment, copper binding agents, and compounds capable of normalizing copper metabolism.

[0002] Incorporation by Reference All U.S. patents, U.S. patent application publications, U.S. patent applications (including provisional patent applications), foreign patents, foreign and PCT published applications, articles and other documents, references and publications described herein, and all cited as references in any patents issued therefrom, are incorporated herein by reference in their entirety. The incorporated information is part of this application as if all text and other content were repeated in this application, and is treated as part of the text and content of this application as filed. [Background technology]

[0003] background The following contains information that may be useful in understanding the present invention. No admission is made that any of the above information is prior art or relevant to any invention described herein or claimed, nor is any admission that any publication or document specifically or implicitly referenced is prior art or a reference that may be used in assessing the patentability of any invention described herein or claimed.

[0004] Essential metals are essential for the normal physiological function of biological systems. They are often present in metalloprotein / metalloenzyme complexes and mediate a variety of processes including mitochondrial function, transcriptional regulation, and cellular metabolism. However, when essential metals are present in concentrations outside the physiological range, they can lead to severe cellular dysfunction.

[0005] Copper is an essential trace element involved in numerous biological processes in living cells. Analysis of the human proteome has identified 54 copper-binding proteins, of which 12 are copper transporters, roughly half are enzymes, and one (antioxidant 1 copper chaperone, ATOX1) is a transcription factor. The majority of copper in the body is found in metabolically active organs such as the liver, kidney, heart, and brain, with approximately 5% of total copper present in serum, up to 95% of which is bound to ceruloplasmin.

[0006] Copper is also involved in oxidative stress: unbound copper behaves as a powerful oxidant, catalyzing the formation of highly reactive hydroxyl radicals, which cause damage to DNA, proteins, and lipids. Therefore, intracellular copper concentrations must be finely regulated by complex homeostatic mechanisms of absorption, excretion, and bioavailability.

[0007] Wilson disease and Menkes disease are genetically transmitted disorders of copper metabolism caused by defects in the genes encoding the ATP-binding cellular copper transporters ATP7B and ATP7A, respectively (de Bie P, et al. Molecular pathogenesis of Wilson and Menkes disease: correlation of mutations with molecular defects and disease phenotypes. J Med Genet 2007;44:673-88). Both excess copper in the brain, such as occurs in Wilson's disease (Cumings JN. The copper and iron content of brain and liver in the normal and in hepato-lenticular degeneration. Brain 1948;71(Pt.4):410-5) and copper deficiency in the brain, such as occurs in Menkes's disease (Walker-Smith JA,et al. Therapeutic implications of copper deficiency in Menkes's steely-hair syndrome.Arch Dis Child 1973;48:958-62), are known to cause severe neurodegeneration if not treated with pharmacological restoration of brain copper levels. Wilson's disease and Menkes disease serve as models for the effects of defective patterns of copper homeostasis on the brain, since it is known that the disease mechanism in each disease is defective copper homeostasis due to copper overload and copper deficiency, respectively, leading to tissue damage.

[0008] Regarding copper and diabetes, there have been some reports of increased copper in urine or serum in people with type 2 diabetes. Cooper, GJ, et al. (2005) Diabetes 54, 1468-147. On the other hand, clinical studies comparing copper levels in plasma or serum of patients with diabetes and healthy individuals have reported conflicting findings (Qiu, Q., et al. Copper in Diabetes Mellitus: a Meta-Analysis and Systematic Review of Plasma and Serum Studies. Biol Trace Elem Res 177, 53-63 (2017)). See also Lowe, J., et al. Dissecting Copper Homeostasis in Diabetes Mellitus. IUBMB Life 69 (4): 255-262 (2017).

[0009] Similarly, although defects in brain metal metabolism occur in various age-related neurodegenerative diseases, there is no consensus regarding the role of such disturbances in metal homeostasis in their pathogenesis. Alzheimer's disease is a neurodegenerative disorder characterized by amyloid plaques in the brain tissue of patients. The plaques are composed mainly of β-amyloid peptides as well as Zn 2+ , Cu 2+ , and Fe 2+Alzheimer's disease is composed of trace elements, including copper, which some have proposed can be considered a form of metal homeostasis disorder, and many studies have focused on copper ions, which appear to be one of the major cationic elements in plaque formation. However, the involvement of copper in Alzheimer's disease is not conclusive, with some studies showing copper deficiency, while other data point to copper overload. Bogheri S.,et al.,Role of Copper in the Onset of Alzheimer's Disease Compared to Other Metals,Front Aging Neurosci.2017;9:446. See Drew,SC,(2017)The Case for Abandoning Therapeutic Chelation of Copper Ions in Alzheimer's Disease,Front.Neurosci.11:317.

[0010] Similarly, many studies have linked increased free copper levels to an increase in Parkinson's disease (another neurodegenerative disease), although this correlation remains unproven. Various studies support the hypothesis that both increased copper (leading to increased intracellular oxidative status) and copper deficiency (leading to the formation of superoxide dismutase 1 aggregates) are correlated with an increased risk of developing the disease. See Marco Bisaglia and Luigi Bubacco, Copper Ions and Parkinson's Disease: Why Is Homeostatsis So Relevant? Biomolecules. 2020 Feb; 10(2): 195. Ajsuvakova OP, et al. Assessment of copper, iron, zinc and manganese status and speciation in patients with Parkinson's disease: A pilot study. J. Trace Elem. Med. Biol. 2019: 126423.

[0011] According to a 2016 study, people with type 2 diabetes are up to 60% more likely to develop another type of dementia, such as Alzheimer's disease or vascular dementia, compared to people without diabetes. Chatterjee S,et al.,Type 2 Diabetes as a Risk Factor for Dementia in Women Compared With Men:A Pooled Analysis of 2.3 Million People Comprising More Than 100,000 Cases of Dementia,Diabetes Care 2016 Feb;39(2):300-307. Some even suggest that Alzheimer's should be classified as a form of diabetes. The term "type 3 diabetes" has been proposed to describe the hypothesis that Alzheimer's disease, the leading cause of dementia, is caused by a type of insulin resistance and insulin-like growth factor dysfunction that occurs specifically in the brain. Some also use this condition to describe people who have type 2 diabetes and are also diagnosed with Alzheimer's dementia.

[0012] There is already an established link between Alzheimer's and type 2 diabetes, according to scientists at the Mayo Clinic, "Researchers Link Alzheimer's Gene to Type 3 Diabetes" (October 25, 2017) www.newsnetwork.mayoclinic.org / discussion / researchers-link-alzheimers-gene-to-type-iii-diabetes, and it has been suggested that Alzheimer's may be caused by insulin resistance in the brain, leading some to say that Alzheimer's is simply "diabetes in the brain." See de la Monte SM and Wands JR, Alzheimer's Disease Is Type 3 Diabetes-Evidence Reviewed, J Diabetes Sci Technol. 2008 Nov;2(6):1101-1113 (concluding that the term "type 3 diabetes" accurately reflects the fact that Alzheimer's disease is a form of diabetes that selectively involves the brain and has molecular and biochemical characteristics that overlap with both type 1 and type 2 diabetes).

[0013] There is strong evidence of an increased incidence of lacunar infarcts and brain atrophy in type 2 diabetes (de Bresser J, et al. Progression of cerebral atrophy and white matter hyperintensities in patients with type 2 diabetes. Diabetes Care 2010;33(6):1309-14; van Harten B, et al. Brain imaging in patients with diabetes:a systematic review. Diabetes Care 2006;29(11):2539-48), when the hippocampus is often severely affected (Gold SM, et al. Hippocampal damage and memory impairments as possible early brain complications of type 2 diabetes. Diabetologia 2007;50(4):711-9; Li M, et al. Atrophy patterns of hippocampal subfields in T2DM patients with cognitive impairment. Endocrine 2020;68:536-48), and even in the early stages (Dong S,et al.Individuals in the prediabetes stage exhibit reduced hippocampal tail volume and executive dysfunction.Brain Behav 2019;9(8):e01351). Gold et al. found deficits in retention of hippocampal-based memory and other cognitive domains. Gold et al. reported that compared with control subjects, individuals with diabetes had reduced brain volume restricted to the hippocampus, and an inverse correlation was found between glycemic control and hippocampal volume.

[0014] In addition to hippocampal atrophy in type 2 diabetes, hippocampal atrophy is also a prominent early stage of neurodegeneration in sporadic Alzheimer's disease, the most common form of dementia (Dubois B,Feldman HH,Jacova C,et al.Advancing research diagnostic criteria for Alzheimer's disease:the IWG-2 criteria.Lancet Neural 2014;13:614-29), in which hippocampal Cu levels are significantly reduced to values ​​similar to those reported in Menkes disease (Walker-Smith JA,et al.Therapeutic implications of copper deficiency in Menkes's steely-hair syndrome.Arch Dis Child 1973;48:958-62). However, copper levels in the cerebrospinal fluid of AD patients are 2.2 times higher than in controls, and elevated ceruloplasmin levels have been found in the brain and cerebrospinal fluid. Basun H., et al.Metals and trace elements in plasma and cerebrospinal fluid in normal aging and Alzheimer's disease.J.Neural.Transm.Park Dis.Dement.Sect.1991;3:231-258.

[0015] Longitudinal epidemiological studies have demonstrated the involvement of chronic hyperglycemia and microvascular disease in the pathogenesis of diabetes-associated cognitive impairment, but the causal pathways underlying the statistical association between type 2 diabetes and dementia are unclear (McCrimmon RJ, et al., Diabetes and cognitive dysfunction. Lancet 2012;379:2291-9).

[0016] The inventors unexpectedly found that copper in the hippocampal region of the brain is significantly elevated in type 2 diabetes, approximating the literature values ​​in Wilson's disease (a neurodegenerative disease of copper excess), while in contrast, the inventors found that hippocampal copper values ​​in the brains of sporadic Alzheimer's disease patients are significantly deficient, similar to Menkes disease (a neurodegenerative disease of copper deficiency).The inventors also found that elevated hippocampal copper levels were the only substantial perturbation of essential metal homeostasis in the brains of patients with type 2 diabetes, with no differences in tissues from the frontal or temporal cortex or meninges. A therapeutic approach is disclosed that reduces copper levels in the hippocampus and is claimed to treat patients with diabetes or vascular dementia who exhibit or are at risk for cognitive impairment, cognitive decline, or who show signs of brain neurodegeneration and / or dementia. [Prior art documents] [Non-patent literature]

[0017] [Non-Patent Document 1] de Bie P, et al.Molecular pathogenesis of Wilson and Menkes disease:correlation of mutations with molecular defects and disease phenotypes.J Med Genet 2007;44:673-88 [Non-Patent Document 2] Cumings JN.The copper and iron content of brain and liver in the normal and in hepato-lenticular degeneration.Brain 1948;71(Pt.4):410-5 [Non-Patent Document 3] Walker-Smith JA,et al.Therapeutic implications of copper deficiency in Menkes' steely-hair syndrome.Arch Dis Child 1973;48:958-62 [Non-Patent Document 4] Cooper, GJ, et al. (2005) Diabetes 54, 1468-147 [Non-Patent Document 5] Qiu,Q.,et al.Copper in Diabetes Mellitus:a Meta-Analysis and Systematic Review of Plasma and Serum Studies.Biol Trace Elem Res 177,53-63(2017) [Non-Patent Document 6] Lowe, J., et al. Dissecting Copper Homeostasis in Diabetes Mellitus. IUBMB Life 69(4):255-262(2017) [Non-Patent Document 7] Bogheri S.,et al.,Role of Copper in the Onset of Alzheimer's Disease Compared to Other Metals,Front Aging Neurosci.2017;9:446 [Non-Patent Document 8] Drew, SC, (2017) The Case for Abandoning Therapeutic Chelation of Copper Ions in Alzheimer's Disease,Front.Neurosci.11:317 [Non-Patent Document 9] Marco Bisaglia and Luigi Bubacco,Copper Ions and Parkinson's Disease:Why Is Homeostatsis So Relevant? Biomolecules.2020 Feb;10(2):195 [Non-Patent Document 10] Ajsuvakova OP,et al.Assessment of copper,iron,zinc and manganese status and speciation in patients with Parkinson's disease:A pilot study.J.Trace Elem.Med.Biol.2019:126423 [Non-Patent Document 11] Chatterjee S,et al.,Type 2 Diabetes as a Risk Factor for Dementia in Women Compared With Men:A Pooled Analysis of 2.3 Million People Comprising More Than 100,000 Cases of Dementia,Diabetes Care 2016 Feb;39(2):300-307 [Non-Patent Document 12] de la Monte SM and Wands JR, Alzheimer's Disease Is Type 3 Diabetes-Evidence Reviewed, J Diabetes Sci Technol. 2008 Nov;2(6):1101-1113 [Non-Patent Document 13] de Bresser J, et al.Progression of cerebral atrophy and white matter hyperintensities in patients with type 2 diabetes.Diabetes Care 2010;33(6):1309-14 [Non-Patent Document 14] van Harten B,et al.Brain imaging in patients with diabetes: a systematic review.Diabetes Care 2006;29(11):2539-48 [Non-Patent Document 15] Gold SM,et al.Hippocampal damage and memory impairments as possible early brain complications of type 2 diabetes.Diabetologia 2007;50(4):711-9 [Non-Patent Document 16] Li M,et al.Atrophy patterns of hippocampal subfields in T2DM patients with cognitive impairment.Endocrine 2020;68:536-48 [Non-Patent Document 17] Dong S,et al.Individuals in the prediabetes stage exhibit reduced hippocampal tail volume and executive dysfunction.Brain Behav 2019;9(8):e01351 [Non-Patent Document 18] Dubois B, Feldman HH, Jacoba C, et al.Advancing research diagnostic criteria for Alzheimer's disease:the IWG-2 criteria.Lancet Neural 2014;13:614-29 [Non-Patent Document 19] Walker-Smith JA,et al.Therapeutic implications of copper deficiency in Menkes' steely-hair syndrome.Arch Dis Child 1973;48:958-62 [Non-Patent Document 20] Basun H.,et al.Metals and trace elements in plasma and cerebrospinal fluid in normal aging and Alzheimer's disease.J.Neural.Transm.Park Dis.Dement.Sect.1991;3:231-258 [Non-Patent Document 21] McCrimmon RJ,et al.,Diabetes and cognitive dysfunction.Lancet 2012;379:2291-9 Summary of the Invention [Means for solving the problem]

[0018] overview The invention described herein and claimed has many attributes and embodiments, including but not limited to those described, illustrated, or referenced in this Summary of the Invention. This Summary of the Invention is not intended to be comprehensive, and the invention described herein and claimed is not limited to or by the features or embodiments identified in this introduction, which is included for purposes of illustration only and not limitation.

[0019] The present invention relates to a method of treating or preventing cognitive impairment, cognitive decline, or symptoms of brain neurodegeneration or dementia in a subject with diabetes, comprising administering to the subject a pharmaceutical composition comprising a compound capable of lowering, lowering, or normalizing copper levels and / or copper metabolism. In some embodiments of the method, the compound is a copper antagonist (such as a copper depleting agent, a copper sequestrant, or a copper remover). In some embodiments, the compound is a copper chelator. In some embodiments, the method of the present invention brings copper levels in the subject within the normal range. In some embodiments, the method of the present invention brings copper levels in the subject within 75-110% of the normal range. In some embodiments, the method of the present invention brings hippocampal copper within the normal expected range, or at least lowers copper within a range that reduces symptoms of cognitive impairment, cognitive decline, or brain neurodegeneration or dementia, or reduces copper. In some embodiments, the method of the present invention is used to bring the blood copper level of the subject to within about 70-140 micrograms per deciliter (mcg / dL). Copper levels may be assessed and monitored, for example, using a urinalysis. Serum and fecal copper measurements are also available, but urinary copper measurements are preferred.

[0020] In some embodiments, a method of treating or preventing cognitive impairment, cognitive decline, or symptoms of brain neurodegeneration or dementia in a subject with diabetes comprises administering to the subject a composition comprising or consisting essentially of a copper chelator or other copper binding compound or copper removing agent. In some embodiments, the composition lowers total copper in the subject. In some embodiments, the composition lowers copper levels in the subject. In some embodiments, the composition lowers the amount of copper(II) and / or copper(I) in the subject. In some embodiments, the composition lowers excess copper(II) and / or copper(I) in the subject. In some embodiments, the composition lowers excess hippocampal copper in the subject. In some embodiments, the excess hippocampal copper in the subject is copper(II). In some embodiments, the excess hippocampal copper in the subject is copper(I). In some embodiments, the composition chelates copper(II) and copper(I) in the subject. In some embodiments of the method, the active ingredient in the composition is trientine. In some embodiments, the trientine is triethylenetetramine disuccinate. In some embodiments, trientine is triethylenetetramine dihydrochloride or triethylenetetramine tetrahydrochloride.

[0021] In some embodiments of the methods, the subject treated in said methods has vascular dementia with or without diabetes, e.g., with or without type 1 diabetes, with or without type 2 diabetes, with or without type 3 diabetes, and / or with or without type 4 diabetes.

[0022] In some embodiments of the method, one or more symptoms of the condition being treated (e.g., cognitive impairment, cognitive decline, or brain neurodegenerative symptoms and / or dementia) are reduced or alleviated. In some embodiments of the method, one or more symptoms of the condition being treated (e.g., cognitive impairment and / or dementia, etc.) are substantially eliminated.

[0023] In some embodiments, the method of the present invention is used for treating frontotemporal dementia (FTD).In some embodiments of the method, one or more symptoms of frontotemporal dementia are reduced or alleviated.In some embodiments of the method, one or more symptoms of frontotemporal dementia are substantially eliminated.

[0024] In some embodiments, the method of the present invention is used for treating frontotemporal dementia (FTLD).In some embodiments of the method, one or more symptoms of frontotemporal dementia are reduced or alleviated.In some embodiments of the method, one or more symptoms of frontotemporal dementia are substantially eliminated.

[0025] In some embodiments, the method of the present invention is used for the treatment of amyotrophic lateral sclerosis (ALS) and / or associated motor neuron disease (MND) dementia.In some embodiments of the method, one or more symptoms of ALS and / or MND are reduced or alleviated.In some embodiments of the method, one or more symptoms of ALS and / or MND are substantially eliminated.

[0026] In some embodiments, the method of the present invention is used for treating dementia with Lewy bodies (DLB).In some embodiments of the method, one or more symptoms of DLB ​​are reduced or alleviated.In some embodiments of the method, one or more symptoms of DLB ​​are substantially eliminated.

[0027] In some embodiments, the method of the present invention is used for the treatment of multiple sclerosis (MS). In some embodiments of the method, one or more symptoms of MS are reduced or alleviated. In some embodiments of the method, one or more symptoms of MS are substantially eliminated.

[0028] In some embodiments, the method of treating FTD, FTLD, ALS, MND, DLB and MS comprises administering to a subject a pharmaceutical composition comprising a compound capable of normalizing copper metabolism.In some embodiments, the compound capable of lowering or lowering copper value, copper level, or total copper, or capable of normalizing copper value, copper level, total copper, or copper metabolism, is a copper chelator or other copper binding or removing agent.Other embodiments of these methods and the agents, compounds, compositions, and procedures useful in these methods are described herein.

[0029] In some embodiments of the method, the subject has diabetes and dementia and / or cognitive decline and / or cognitive impairment and / or memory impairment. In some embodiments, the subject has type 2 diabetes. In other embodiments, the subject has type 1 diabetes. In some embodiments, the subject has type 3 diabetes. In some embodiments, the subject has type 4 diabetes.

[0030] In some embodiments, the subject being treated has type 1 diabetes, and one or more of the cognitive domains negatively affected by the cognitive impairment in the subject are reduced overall cognition, fluid and crystallized intelligence, information processing speed, psychomotor efficiency, visual and sustained attention, mental flexibility, and / or vision.

[0031] In some embodiments, the subject being treated has type 2 diabetes, and one or more of the cognitive domains negatively affected by the cognitive impairment in the subject are memory (verbal memory, visual retention, working memory, immediate recall, delayed recall), psychomotor speed and frontal / executive function, processing speed, complex motor function, verbal fluency, and / or attention.

[0032] In some embodiments, cognitive impairment interferes with concentration, memory recall, and / or leads to mental fatigue in individuals with diabetes (eg, type 2 diabetes). In some embodiments of the method of the present invention, the structural correlation of diabetes-related cognitive impairment is assessed using brain magnetic resonance imaging (MRI).MRI can be used to evaluate subjects before and / or during treatment.In some embodiments, in the evaluation of cognitive impairment in patients with diabetes (e.g., patients with type 2 diabetes) for the treatment described herein, MRI is used to test or confirm the presence of normal or abnormal brain structure before and / or during treatment.

[0033] In some embodiments, in patients with type 2 diabetes, for example, white matter hyperintensities correlate with impaired performance on tests of attention, executive function, information processing speed, and memory, providing a structural rationale for treatment with the compounds of the invention. In some embodiments, MRI is used to test for or confirm the presence of white matter hyperintensities before and / or during treatment.

[0034] In some embodiments, MRI is used to demonstrate or confirm that a subject with diabetes (e.g., type 2 diabetes) has lacunar infarct(s), hippocampal and / or amygdala atrophy or other hippocampal deficits (including hippocampal atrophy patterns) prior to treatment with a compound of the invention. In some embodiments, MRI is used to evaluate a subject with diabetes (e.g., type 2 diabetes) for hippocampal and / or amygdala atrophy or other hippocampal deficits (including hippocampal atrophy patterns) during treatment with a compound of the invention.

[0035] In some embodiments, MRI is used to assess hippocampal and / or amygdala atrophy in subjects with type 2 diabetes before, during, and / or after treatment according to the methods of the present invention.

[0036] In some embodiments, the present invention relates to a method for treating or preventing cognitive impairment in a subject with vascular dementia, sometimes referred to as vascular cognitive impairment or VCI, comprising administering to the subject a pharmaceutical composition comprising a compound capable of normalizing copper metabolism. In some embodiments, the subject may or may not have diabetes, for example, may or may not have type 1 diabetes, may or may not have type 2 diabetes, may or may not have type 3 diabetes, or may or may not have type 4 diabetes. In some embodiments, the compound capable of lowering or lowering copper levels, copper levels, or total copper or capable of normalizing copper levels, copper levels, total copper, or copper metabolism is a copper chelator or other copper binding or copper removing agent. In some embodiments, the subject with vascular dementia or VCI has problems with reasoning, planning, judgment, memory, and other thought processes. In some embodiments, the composition lowers total copper in the subject. In some embodiments, the composition lowers copper levels in the subject. In some embodiments, the composition reduces the amount of copper(II) and / or copper(I) in the subject. In some embodiments, the composition reduces excess copper(II) and / or copper(I) in the subject. In some embodiments, the composition reduces excess hippocampal copper in the subject. In some embodiments, the excess hippocampal copper in the subject is copper(II). In some embodiments, the excess hippocampal copper in the subject is copper(I). In some embodiments, the composition chelates copper(II) and / or copper(I) in the subject. In some embodiments, the active ingredient in the composition is trientine. In some embodiments, the trientine is triethylenetetramine disuccinate, triethylenetetramine dihydrochloride, and / or triethylenetetramine tetrahydrochloride.

[0037] In some embodiments of the methods of the present invention, the compound capable of normalizing copper metabolism is capable of lowering or reducing elevated copper in a subject.

[0038] In some embodiments of the methods of the invention, the copper antagonist (e.g., a copper lowering / removing or copper normalizing compound) in the composition is a copper antagonist. 2+ In some embodiments, the copper antagonist compound in the composition binds to copper. 2+ In some embodiments of the methods of the present invention, the copper removing agent in the composition administered to the subject is copper. 2+ thereby lowering total copper, copper levels, and / or hippocampal copper in the subject.

[0039] In some embodiments of the method of the present invention, the administered compound effective for lowering total copper or copper values ​​content in the subject is a copper chelating compound. In some embodiments, the administered compound effective for lowering total copper or copper values ​​content in the subject comprises, consists essentially of, or consists of an agent that binds or chels copper(I). In other embodiments, the administered compound effective for lowering total copper or copper values ​​content in the subject comprises, consists essentially of, or consists of an agent that binds or chels copper(II). In other embodiments, the administered agent effective for lowering total copper or copper values ​​content in the subject comprises, consists essentially of, or consists of an agent that binds or chels both copper(I) and copper(II).

[0040] In one embodiment, the compounds effective to reduce copper or lower total copper or lower copper content in a subject, resulting in hippocampal copper, include D-penicillamine; N-acetylpenicillamine; triethylenetetramine (also known as TETA, TECZA, triene, triene, and trientine) and pharma- ceutically acceptable salts thereof; trithiomolybdate, tetrathiomolybdate, ammonium tetrathiomolybdate, choline tetrathiomolybdate; bis-choline tetrathiomolybdate (thiomolybdate USAN, trade name Decuprate), 2,2,2 tetramine tetrahydrochloride; 2,3,2 tetramine tetrahydrochloride; ethyl tetrathiomolybdate; diaminetetraacetate (EDTA, a non-preferential, non-specific metal binder that is dosed with caution to avoid toxicity); diethylenetriaminetetraacetic acid (DPTA, a non-preferential, non-specific metal binder that is dosed with caution to avoid toxicity due to chelation of essential metals (such as Zn and Mn); 5,7,7'12,14,14'hexakismethyl-1,4,8,11 tetraazacyclotretradecane; 1,4,8,11 tetraazacyclotretradecane, e.g., cyclam S, cyclamus, and copper-chelating cyclam derivatives, e.g., Bn-cyclam-EtOH, oxo-cyclam-EtOH and oxo-Bn-cyclam-EtOH, (HOCH 2 CH 2 CH 2 ) 2 (PhCH 2 ) 2 Cyclam, and [ka] ;1,4,8,11-Tetraazabicyclotetradecane-1,4,8,11-tetraacetic acid;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;Melatonin;Cyclic 3-hydroxymelatonin (3OHM);N(1)-Acetyl-N(2)-formyl-5-methoxykynuramine (AFMK);N(1)-Acetyl-5-methoxykynuramine (AMK);N,N'-Diethyldithiocarbamate;Bathocuproinedi The compound or agent for reducing the amount and / or level of copper, reducing total copper, or reducing copper value content comprises, consists essentially of, or consists of a compound selected from the group consisting of: sulfonic acid; bathocuprine disulfonate; trimetazidine; triethylenetetramine tetrahydrochloride; 2,3,2-tetraamine; 1,10-orthophenanthroline; 3,4-dihydroxybenzoic acid; 2,2'-bicinchoninic acid; diamsar; 3,4',5, trihydroxystilbene (resveratrol); mercaptodextran; disulfiram (Antabuse); sarcofagin; DiAmSar; diethylenetriaminepentaacetic acid; and diethylenetriaminepentaacetic acid calcium trisodium; neocuproine; bathocuproine; and carnosine. In some embodiments, the compound or agent for reducing the amount and / or level of copper, reducing total copper, or reducing copper value content comprises Cu 1+ In another embodiment, the compound or agent for reducing the amount and / or level of copper, reducing total copper, or reducing copper content is Cu 2+ In some embodiments, Cu 2+ The compound or agent for reducing the amount and / or level of copper, reducing total copper, or reducing copper content that preferentially binds to Cu is triethylenetetramine disuccinate. 1+ and Cu 2+ In some embodiments, Cu binds to both 1+ and Cu 2+A compound or agent for reducing the amount and / or level of copper, reducing total copper, or reducing copper content that preferentially binds to both is a penicillamine copper chelator. In some embodiments, the penicillamine copper chelator is D-penicillamine.

[0041] In some embodiments, the agent is administered to reduce total and / or hippocampal copper in the subject.In some embodiments, the pharmaceutical composition used in the method of the present invention comprises a therapeutically effective amount of triethylenetetramine and a pharma- ceutically acceptable carrier, glidant, diluent, or excipient.In some embodiments, the triethylenetetramine is in the form of a pharma- ceutically acceptable salt.

[0042] In another embodiment, administration of a compound or agent for reducing total copper and / or hippocampal copper maintains total copper in the subject within the range of normal human serum or plasma of about 0.8-1.2 milligrams / L, or about 10-25 micromoles / L. In another embodiment, the agent for reducing copper maintains total copper in the subject within at least about 70% (e.g., at least about 75%) of the normal range of about 0.8-1.2 milligrams / L or about 10-25 micromoles / L. In another embodiment, the compound or agent for reducing copper maintains total copper in the subject within about 75% to about 85%, or about 85% to about 95% of the normal range of copper in human plasma or serum. In one aspect of the method of the present invention, the copper status of the subject provided by the compound or agent for reducing copper is determined by assessing copper in the subject's urine. In some embodiments, the copper status of the subject provided by the compound or agent for reducing copper is determined by assessing copper in the subject's plasma. In some embodiments, the copper status of a subject provided by a copper reducing compound or agent is determined by assessing copper in the liver of the subject.

[0043] In one embodiment, triethylenetetramine is the hydrochloride salt of triethylenetetramine. In some embodiments, triethylenetetramine is the succinate salt of triethylenetetramine.

[0044] In one embodiment of the invention, the method employs a pharmaceutical composition comprising substantially pure triethylenetetramine disuccinate and a pharma- ceutically acceptable excipient. In another embodiment, the method employs a pharmaceutical composition comprising substantially pure triethylenetetramine dihydrochloride and / or tetrahydrochloride and a pharma- ceutically acceptable excipient.

[0045] In one embodiment of the invention, the method employs a pharmaceutical composition comprising a crystalline form of triethylenetetramine disuccinate or a crystalline form of the hydrochloride salt of triethylenetetramine.In another embodiment of the invention, the method employs a pharmaceutical composition comprising triethylenetetramine disuccinate anhydrate or the hydrochloride salt of triethylenetetramine anhydrate.

[0046] In certain embodiments, the triethylenetetramine succinate is a triethylenetetramine disuccinate polymorph. In certain embodiments, the triethylenetetramine hydrochloride is a triethylenetetramine hydrochloride polymorph.

[0047] In some embodiments of the method of the present invention, the method comprises administering to a subject a therapeutically effective amount of a compound selected from the group consisting of trientine, succinic acid addition salt of triethylenetetramine, hydrochloric acid addition salt of triethylenetetramine, and pharmaceutically acceptable salts of D-penicillamine, N-acetylpenicillamine, tetrathiomolybdate, ammonium tetrathiomolybdate, and choline tetrathiomolybdate.The compound reduces total copper, copper level, and / or hippocampal copper in the subject treated as described herein, and one or more symptoms of cognitive impairment, cognitive decline, and / or dementia, including vascular dementia.The reduction in total copper or copper level in the subject results in and substitutes for the reduction in hippocampal copper.

[0048] In some embodiments of the method of the present invention, the subject shows signs of brain degeneration or cognitive impairment before treatment. In other embodiments, the subject has or is at risk of brain degeneration or cognitive impairment, or is at risk of cognitive decline and / or dementia. The risk for these conditions can be determined by measuring copper levels in the subject. In some embodiments, copper is measured in the urine of the subject before treatment, during treatment, or both. Another risk factor is a diagnosis of diabetes with shrinkage of the hippocampus and / or amygdala as shown by MRI or reduced glucose uptake in the hippocampus and / or amygdala as shown by positron emission tomography (PET) scan.

[0049] In some embodiments, subjects with diabetes or vascular dementia have elevated urinary copper excretion. In some embodiments, subjects with diabetes or vascular dementia have impaired spatial memory or the ability to remember directions, locations, and orientation. In some embodiments, subjects with diabetes or vascular dementia have elevated urinary copper excretion and impaired spatial memory or the ability to remember directions, locations, and orientation.

[0050] In some embodiments of the methods of the present invention, the methods further comprise administering one or more additional therapeutic agents selected from an anti-inflammatory agent, an agent for treating cardiovascular disease, an agent for treating hypertension, an agent for treating kidney disease, an agent for treating depression, and an agent for treating type 2 diabetes and / or dementia.

[0051] In some embodiments, the one or more additional therapeutic agents for treating type 2 diabetes are selected from the group consisting of alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 receptor agonists, meglitinides, sodium glucose transporter (SGLT) 2 inhibitors, sulfonylureas, and thiazolidinediones.

[0052] In some embodiments, the one or more additional therapeutic agents for treating dementia are selected from the group consisting of cholinesterase inhibitors, antibodies targeting amyloid beta protein (a biomarker for Alzheimer's disease and other dementias), and N-methyl-D-aspartate (NMDA) receptor antagonists. In some embodiments, the cholinesterase inhibitor is donepezil (Aricept), galantamine (Razadyne, Razadyne ER, Reminyl), or Rivastigmine (Exelon). In some embodiments, the antibody targeting amyloid beta protein is Aducanumab-avwa (Aduhelm). In some embodiments, the NMDA receptor antagonist is memantine (Axura, Ebixa, Namenda, etc.).

[0053] In some embodiments, the subject is a human.

[0054] In some embodiments, the pharmaceutical composition is administered orally in the form of a capsule or tablet.

[0055] In some embodiments, the compound is triethylenetetramine dihydrochloride and is administered in an amount of about 1200 mg daily. In some embodiments, 1200 mg of triethylenetetramine dihydrochloride is administered in 600 mg divided doses BID, in 400 mg divided doses TID, or in 300 mg divided doses QID.

[0056] In some embodiments, the compound is triethylenetetramine disuccinate and is administered in a dose ranging from about 2400 mg / day to about 3000 mg / day or more. In some embodiments, the compound is triethylenetetramine disuccinate and is administered in an amount of about 2800 mg / day. Other useful doses of triethylenetetramine disuccinate are administered, such as about 1050 mg / day to about 2300 mg / day, about 1400 mg / day to about 3500 mg / day, about 2400 mg / day to about 3200 mg / day, and about 2800 mg / day to about 5600 mg / day. In some embodiments, these daily amounts of triethylenetetramine disuccinate are administered in divided doses.

[0057] The present invention also provides a kit for therapeutic treatment of treating or preventing cognitive impairment in a subject with diabetes, comprising: a) a pharmaceutical composition comprising a copper antagonist compound (comprising one or more of the compounds described herein, including copper(I) and / or copper(II) chelators) capable of lowering or lowering copper (e.g., lowering or lowering copper levels, lowering or lowering total copper, lowering or lowering copper levels and / or amounts (e.g., urinary copper levels and / or amounts)) and / or normalizing copper metabolism in a subject; and b) instructions for use in therapeutic treatment or prevention of cognitive impairment, cognitive decline and / or dementia, etc., as described herein, in a subject with diabetes. In some embodiments, the subject has type 1, type 2, type 3, or type 4 diabetes. In some embodiments, the subject of the instructions has vascular dementia, with or without diabetes.

[0058] In some embodiments, the compound in the kit is selected from the group consisting of triethylenetetramine dihydrochloride, triethylenetetramine tetrahydrochloride, and triethylenetetramine disuccinate. In some embodiments, the triethylenetetramine disuccinate in the kit is triethylenetetramine disuccinate anhydride.

[0059] In one embodiment, the invention includes an article of manufacture comprising a package insert instructing a user to administer a copper antagonist compound (comprising one or more of the compounds described herein that include copper(I) and / or copper(II) chelators) capable of lowering or lowering copper (e.g., lowering or lowering copper levels, lowering or lowering total copper, and / or normalizing copper metabolism) in a subject to a patient having a condition or disorder characterized by diabetes and dementia (or risk of dementia). The condition or disorder characterized by dementia in a patient with diabetes can be Alzheimer's disease. The condition or disorder characterized by dementia in a patient with diabetes can be vascular dementia.

[0060] In a further aspect, the comorbidity in dementia patients (or patients at risk of dementia) treatable with a compound capable of normalizing copper metabolism (e.g., one or more of the copper chelators described herein) is characterized by excess copper (e.g., type 2 diabetes). In a further aspect, the comorbidity in dementia patients treatable with a compound capable of normalizing copper metabolism (e.g., one or more of the copper chelators described herein) is characterized by copper deficiency (e.g., Alzheimer's disease). In another aspect, the disease, condition, or disorder is selected from the group consisting of diabetes, Alzheimer's disease, and Parkinson's disease.

[0061] A preferred pharmaceutical composition for use in the methods of the present invention comprises, consists essentially of, or consists of substantially pure triethylenetetramine disuccinate. Another preferred composition comprises, consists essentially of, or consists of substantially pure triethylenetetramine disuccinate anhydride. Another preferred composition is a composition that comprises, consists essentially of, or consists of substantially pure triethylenetetramine disuccinate crystals having alternating layers of triethylenetetramine and succinate molecules.

[0062] In another aspect of the invention, the methods of the invention maintain copper levels at about 70% to about 110% of normal levels in a subject, thereby lowering copper levels in a mammalian patient and / or lowering or inducing lowering of hippocampal copper levels.

[0063] Both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further details of the invention and are not to be construed as limiting. Other objects, advantages, and novel features will become apparent to those skilled in the art from the following detailed description of the invention. [Brief description of the drawings]

[0064] [Figure 1] Figure 1 shows concentrations of nine essential elements (A-I) in four human brain regions comparing control (red) and T2D (green) postmortem tissue. Data are means ± 95% Cl. For MN data, single outliers derived from both Mg and Cu analyses were removed from the plots for clarity: this did not change the results or conclusions. Nonstandard abbreviations: FC, frontal cortex; TC, temporal cortex; HP, hippocampus; MN, meninges.

[0065] [Diagram 2] Figure 2 shows data from a second technical replicate analysis corresponding to Figure 1, showing replicate measurements of dry weight concentrations of nine essential elements (A-I) in four human brain regions comparing control (red) and T2D (green) human postmortem tissue. Data are mean ± 95% CI. Within the MN, single outliers derived from Mg and Cu were removed from the plot for clarity of presentation: this did not change the results or conclusions. Nonstandard abbreviations: FC: frontal cortex; TC: temporal cortex; HP: hippocampus; MN: meninges.

[0066] [Figure 3A]Figure 3 shows two-dimensional PCA and PLS-DA plots with VIP scores for human dry weight hippocampal postmortem tissue. Data represent A) PCA plots and B) PLS-DA plots for AD (red; n=9), control (green; n=14), and T2D (blue; n=6) hippocampal tissue. Colored ellipses for PCA and PLS-DA plots represent 95% confidence intervals. In the PCA plot, the first principal component (PCI) represents 48.3% of the total variance, whereas the second (PC2) accounts for 19.3%. In both the PCA and PLS-DA plots, the two cases (AD vs. T2D) demonstrate almost complete separation, whereas there is considerable overlap between T2D and controls. Plots of VIP scores (bottom) show the relative contribution of each metal to the variance between AD, T2D, and controls in C) component 1 and D) component 2. A higher VIP score indicates a higher contribution to the separation of the groups. Colored boxes on the right indicate whether metal concentrations are increased (red) or decreased (green) in the diseased group. For both components, Cu, Na, and Mn achieved the top three VIP scores, with Na achieving the highest VIP score in both components. Nonstandard abbreviations: AD: Alzheimer's disease; Con: Control; PCA: Principal Component Analysis; PC: Principal Component; PLS-DA: Partial Least Squares-Discriminant Analysis; T2D: Type 2 Diabetes; VIP: Variable Importance in Projection. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above.

[0067] [Figure 4] Figure 4 shows a scree plot for dry weight hippocampal postmortem tissue. Data represents a scree plot showing the individual variance explained for the top five dimensions following hippocampal PCA.

[0068] [Figure 5A]Figure 5 shows two-dimensional PCA and PLS-DA plots for human dry weight T2D temporal cortex and AD temporal gyrus postmortem tissue. Data are presented in Figure 5(A) PCA and Figure 5(B) PLS-DA plots for AD (red; n=9; mineral temporal gyrus), controls (green; n=13; temporal cortex / middle temporal gyrus), and T2D (blue; n=6: temporal cortex). Colored ellipses for PCA and PLS-DA plots represent 95% confidence intervals. Furthermore, in the PCA plot, the first principal component (PC1) represents 41.2% of the total variance, whereas the second (PC2) accounts for 24.5%. The two cases (AD vs. T2D) show no separation in the PCA plot, whereas in the PLS-DA plot, separation is evident due to the incorporation of supervised modeling. [Figure 5B] Same as above.

[0069] [Figure 6A] Figure 6 shows two-dimensional PCA and PLS-DA plots for human dry weight frontal cortex and meningeal postmortem tissue. Data represent PCA (Figures 6A and 6C) and PLS-DA (Figures 6B and 6C) plots for control (red; n=6) and T2D (green; n=6) frontal cortex (Figures 6A and 6B) and meningeal (Figures 6C and 6D) tissue. Colored ellipses for PCA and PLS-DA plots represent 95% confidence intervals. No separation was observed in any multivariate plot for both frontal cortex and meningeal postmortem tissue. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Detailed Description Both diabetes and dementia are prevalent among older adults. The risk of dementia is increased in patients with diabetes, and patients with dementia and diabetes appear to be at higher risk for severe hypoglycemia. The exact factors that contribute to the increased dementia risk in older adults with diabetes are not known. Neurobiological changes seen in the aging hippocampus, including increased oxidative stress and neuroinflammation, altered intracellular signaling and gene expression, and reduced neurogenesis and synaptic plasticity, are thought to be associated with age-related cognitive decline. Diabetes (including type 2 diabetes) has been shown to increase the risk of cognitive decline and dementia, for example in Alzheimer's disease and vascular dementia.

[0071] The adverse effects of diabetes on the retina, kidney, cardiovascular system, and peripheral nervous system are widely recognized. Less attention has been paid to the effects of diabetes on cognitive function. Both type 1 and type 2 diabetes are associated with impaired performance in multiple cognitive domains. The exact pathophysiology of cognitive dysfunction in diabetes is not fully understood, but it is believed that hyperglycemia, vascular disease, hypoglycemia, and insulin resistance may play important roles. Modalities for studying the effects of diabetes on the brain, including neurocognitive tests, evoked response potentials, and magnetic resonance imaging, have evolved over the years and are useful in conjunction with the methods of the present invention both before and during treatment.

[0072] A recent meta-analysis included 33 studies that tested cognitive function in adult subjects with diabetes. Kodl and Seaquist,Cognitive Dysfunction and Diabetes,Endocr Rev.2008 Jun;29(4):494-511. Significant declines in general cognition, fluid and crystallized intelligence, information processing speed, psychomotor efficiency, visual and sustained attention, mental flexibility, and vision were found in subjects with type 1 diabetes compared with controls. There were no differences in memory, motor speed, selective attention, or language. (See Table 1 in Kodl and Seaquist.)

[0073] Kodl and Seaquist report in their paper that patients with type 2 diabetes were also found to have cognitive impairment (see Table 2 in Kodl and Seaquist, ibidem). The authors provide citations to show that type 2 diabetes is associated with reduced psychomotor speed, frontal / executive function, verbal memory, processing speed, complex motor function, working memory, immediate recall, delayed recall, verbal fluency, visual retention, and attention. They refer to a study by Sinclair et al. (2000 Cognitive dysfunction in older subjects with diabetes mellitus: impact on diabetes self-management and use of care services. All Wales Research into Elderly (AWARE) Study. Diabetes Res Clin Pract 50:203-212) which found that subjects who scored less than 23 on the Mini-Mental Status Examination fared worse on measures of self-care and ability to perform activities of daily living compared to controls, and also had increased personal care needs and increased hospitalization rates. Type 2 patients also have an increased incidence of Alzheimer's disease and an increased incidence of vascular dementia. Recently, Bruce et al. found that 17.5% of older patients with type 2 diabetes had moderate to severe deficits in activities of daily living, 11.3% had cognitive impairment, and 14.2% had depression (2003 Cognitive impairment, physical disability and depressive symptoms in older diabetic patients: the Fremantle Cognition in Diabetes Study. Diabetes Res Clin Pract 61:59-67). See also Zillox L., et al., Diabetes and Cognitive Impairment, Curr Diab Rep. 2016 Sept;16(9):87.

[0074] Type 2 diabetes is characterized by chronic hyperglycemia and a propensity for glucose-mediated damage in multiple organs, impaired copper (Cu) homeostasis, elevated rates of cognitive impairment and dementia, and an epidemiological association with sporadic Alzheimer's disease (sAD). In contrast, sAD exhibits widespread progressive age-related brain neurodegeneration and dementia, widespread elevation of brain glucose without significant hyperglycemia, and widespread brain copper deficiency.

[0075] The following example study describes a method to determine whether sAD-like brain metal fluctuations occur in T2D, in which the levels of nine essential metals are measured from four brain regions in cases with short postmortem delay (n=6) and matched controls (n=6) and compared with case-control data from a study of sAD (n=9 / group) performed with equivalent methodology. Both supervised and unsupervised multivariate statistical methods are used to contrast group differences in hippocampal data. To determine whether sAD-like brain metal fluctuations occur in type 2 diabetes, the levels of nine essential metals are measured from four brain regions in cases with short postmortem delay (n=6) and matched controls (n=6) and compared with case-control data from a study of sAD (n=9 / group) performed with equivalent methodology. Both supervised and unsupervised multivariate statistical methods are used to contrast group differences in hippocampal data.

[0076] Unexpectedly, we found that hippocampal Cu levels were significantly elevated in T2D ( P = 0.005 and 0.007 for consecutive technical replicates) and approximated literature values ​​in Wilson's disease (WD) brains, whereas, in contrast, hippocampal Cu values ​​were significantly depleted in sAD. Multivariate analysis identified striking differences in corresponding metal-associated patterns between hippocampal case-control datasets derived from type 2 diabetes and sAD.

[0077] In other words, the hippocampal copper level in the brain is significantly elevated in type 2 diabetes, while the hippocampal copper level in the brain of sporadic Alzheimer's disease patients is significantly deficient. It is understood that failure to remove excess copper in the WD brain may cause irreversible brain damage.

[0078] The hippocampus is often affected early in the development of both type 2 diabetes and sAD-induced neurodegeneration. As shown in the following examples, hippocampal Cu levels were significantly elevated in type 2 diabetes cases, whereas in contrast, hippocampal Cu was deficient in sAD, which is consistent with the reported severe and widespread brain Cu deficiency. Thus, the mechanism of hippocampal Cu changes is different between type 2 diabetes and sAD. Elevated hippocampal Cu is believed to contribute to the pathogenesis of brain neurodegeneration and cognitive impairment in type 2 diabetes, consistent with the known deleterious effects of similar elevated brain Cu in WD. In patients with type 2 diabetes who show signs of cognitive impairment or brain neurodegeneration, such as those described herein, therapeutic brain copper lowering approaches similar to those currently used in WD would be useful.

[0079] With regard to the known deleterious effects of elevated brain copper in WD, elevated hippocampal Cu levels are consistent with its contribution to the pathogenesis of brain neurodegeneration and cognitive impairment in type 2 diabetes. Therapeutic approaches to lower hippocampal and brain copper would be useful in patients with type 2 diabetes who show signs of or are at risk for cognitive impairment or brain neurodegeneration, or who test positive after elevated hippocampal and / or brain copper.

[0080] The following examples show that elevated hippocampal Cu levels were the only substantial perturbation in essential metal homeostasis detected in the brains of patients with type 2 diabetes. In contrast, there were no substantial differences between cases and controls in tissues from the frontal or temporal cortex, or the meninges.

[0081] To further determine the significance of these findings, an additional analysis was performed to contrast the observations in this case-control study of type 2 diabetes with individual patient measurements derived from a directly comparable case-control study of sAD. In this analysis, brain metal levels were measured by the same method as used herein for type 2 diabetes and control subjects. Unexpectedly, in direct contrast to the findings in type 2 diabetes, hippocampal Cu levels in sAD were substantially lower than matched controls. Thus, the pattern of Cu dysregulation measured in sAD (i.e., hippocampal Cu deficiency) was in stark contrast to the pattern in type 2 diabetes (hippocampal Cu elevation). Thus, hippocampal Cu levels are elevated in type 2 diabetes compared to control values, whereas in sAD they are decreased compared to controls. Furthermore, Cu fluctuations in sAD are widespread, whereas in type 2 diabetes the fluctuations may be more limited. It can be concluded that the processes that result in or cause the more limited defects in Cu homeostasis in type 2 diabetes are fundamentally different from those that cause the widespread brain Cu deficiency in sAD.

[0082] Cu is the third most abundant transition metal in the brain (after Zn and Fe) and plays a key role in many processes including cellular respiration, regulation of Fe metabolism, and antioxidant pathways. Defects in copper regulation are known to play a central role in the pathogenesis of two genetic disorders: WD, characterized by toxic copper overload in the liver, eyes, and brain, and conversely, Menkes disease, in which brain damage is caused by cerebral copper deficiency. Both of these diseases can lead to severe neurodegeneration unless normal brain copper levels are restored by prompt pharmacological intervention after diagnosis. Here, we confirmed that the increase in Cu levels measured in the T2D hippocampus approximated the fold changes in regions adjacent to the hippocampus as reported for WD (Table 3).

[0083] Our root cause analysis (RCA) revealed that signals from sAD and type 2 diabetes were virtually completely separated, whereas type 2 diabetes and control hippocampal tissues showed substantial overlap. This is in contrast to parallel findings in other type 2 diabetes brain regions studied, where case and control values ​​were substantially overlapping. To further test discrimination between cases and controls, we employed PLS-DA (partial least squares-discriminant analysis) modeling, which showed similar cluster separation to the RCA analysis. These findings further reveal contrasting patterns of brain metal homeostasis abnormalities between type 2 diabetes and sAD. Based on PLS-DA modeling, the VIP scores revealed that Na, Mn, Cu, and Fe each had a score above 1, indicating that these metals provide a reliable discriminant for cluster separation. Na achieved the top VIP score in both components, whereas Cu ranked third and second for VIP scores in components 1 and 2, respectively. This indicates that Cu is one of the key variables responsible for the separation of AD and type 2 diabetes clustering, supporting the conclusion that Cu variation plays a key role in type 2 diabetes neuropathogenesis. Mirroring our initial ICP-MS findings, identification of metal dysregulation using the same multivariate analysis was not evident between type 2 diabetes and controls in the remaining brain regions. Although shared pathophysiological characteristics have been previously observed between AD and type 2 diabetes (Chatterjee S, Mudher A. Alzheimer's disease and type 2 diabetes: A critical assessment of the shared pathological traits. Front Neurosci 2018 Jun 8;12:383), these findings support the possibility of contrasting Cu-related neurodegenerative processes in these two diseases.

[0084] To explore the possible effect of sample size in this study (T2D vs. control, n=6 / 6), a post-hoc statistical power test of hippocampal tissue was performed to ensure the absence of type II error. Across the two technical replicates, Cu had the highest power level (>0.90, see Table 7 in the Examples below), whereas all other metals had power levels below 0.80. Furthermore, a priori analysis found that only Cu had a desirable sample size limit (n=10) below that used in this study. These power levels are taken into account when interpreting differences in other hippocampal metal levels measured. A recent paper reported that brain metal levels were not affected by the length of PMD up to 72 hours. Therefore, it can also be concluded that PMD in this study did not significantly affect brain metal levels. See Scholefield M, et al.Evidence that levels of nine essential metals in postmortem human-Alzheimer's-brain and ex vivo rat-brain tissues are unaffected by differences in postmortem delay, age, disease staging, and brain bank location.Metallomics 2020;12:952-62.

[0085] In summary, the studies in the examples provide solid evidence that hippocampal Cu levels are elevated in cases of type 2 diabetes, whereas hippocampal Cu was correspondingly decreased in sAD. This is the first study to report metal concentrations in multiple regions in the brain of type 2 diabetes using a metallomic method that can simultaneously quantitatively measure all essential metals in a manner that cannot be reached by other methodologies such as MRI. The fold changes of Cu in type 2 diabetes approximated those reported in WD, where neurodegeneration is due to Cu toxicity. Using PCA and PLS-DA, substantially contrasting patterns of brain metal levels were identified between type 2 diabetes, sAD, and controls. Cu had the highest VIP score, indicating it was the key discriminant responsible for the separation between sAD and type 2 diabetes clustering here. Taken together, these findings demonstrate that contrasting neurodegenerative processes exist in type 2 diabetes and sAD.

[0086] Therapeutic copper lowering and copper normalization approaches are disclosed and claimed for treating patients with diabetes who show or are at risk of cognitive impairment, cognitive decline and / or dementia, or who show signs of brain neurodegeneration.The methods of the present invention are also useful for prophylactic treatment or prevention of cognitive impairment, cognitive decline and / or dementia, etc. in patients with diabetes.The patients include patients with diabetes (e.g., type 2 diabetes), and / or vascular dementia with or without diabetes.The methods of the present invention are also useful for lowering chelatable copper in these subjects to treat or prevent these conditions.

[0087] In some embodiments, therapeutic copper lowering and copper normalization approaches are disclosed and claimed for treating (both proactively and preventatively) patients with, suspected of, or at risk of having frontotemporal dementia (FTD).In some method embodiments, one or more symptoms of frontotemporal dementia are reduced or alleviated.In some method embodiments, one or more symptoms of frontotemporal dementia are substantially eliminated.

[0088] In some embodiments, therapeutic copper lowering and copper normalization approaches are disclosed and claimed for treating (both proactively and preventatively) patients with, suspected of, or at risk of having frontotemporal dementia (FTLD).In some method embodiments, one or more symptoms of frontotemporal dementia are reduced or alleviated.In some method embodiments, one or more symptoms of frontotemporal dementia are substantially eliminated.

[0089] In some embodiments, therapeutic copper lowering and copper normalization approaches are disclosed and claimed for treating (both proactively and preventatively) patients with, suspected of, or at risk of having amyotrophic lateral sclerosis (ALS) and / or associated motor neuron disease (MND) dementia. In some method embodiments, one or more symptoms of ALS and / or MND are reduced or alleviated. In some method embodiments, one or more symptoms of ALS and / or MND are substantially eliminated.

[0090] In some embodiments, therapeutic copper lowering and copper normalization approaches are disclosed and claimed for treating (both proactively and preventively) patients with, suspected of, or at risk of having dementia with Lewy bodies (DLB).In some method embodiments, one or more symptoms of DLB ​​are reduced or alleviated.In some method embodiments, one or more symptoms of DLB ​​are substantially eliminated.

[0091] In some embodiments, therapeutic copper lowering and copper normalization approaches are disclosed and claimed for treating (both proactively and preventatively) patients with, suspected of, or at risk of having multiple sclerosis (MS). In some method embodiments, one or more symptoms of MS are reduced or alleviated. In some method embodiments, one or more symptoms of MS are substantially eliminated.

[0092] In some embodiments, the method for treating FTD, FTLD, ALS, MND, DLB, and / or MS comprises or consists essentially of administering to a subject a pharmaceutical composition comprising a compound capable of normalizing copper metabolism.In some embodiments, the compound capable of lowering or lowering copper value, copper level, or total copper, or capable of normalizing copper value, copper level, total copper, or copper metabolism, is a copper chelator or other copper binding agent or copper remover.Other embodiments of these methods for treating FTD, FTLD, ALS, MND, DLB, and / or MS, as well as the agents, compounds, compositions, and procedures useful in these methods are described herein (including, for example, the copper antagonists, copper chelators, and compounds useful in treating the above-mentioned diseases and disorders, capable of normalizing copper value).

[0093] definition Copper(II) referred to herein is Cu(II) or Cu +2 Or copper +2 or "copper(II)" +2 Copper(I) as referred to herein is either Cu(I) or Cu +1 Or copper + or as "copper(I)" +1 These are also known as cations.

[0094] The term "chelatable copper" as used herein includes copper in any of its chelatable forms, including different oxidation states such as copper(I) and copper(II). Thus, the term "copper level" (e.g., elemental, salt, etc.) refers to any suitable form of copper in the body that is available for such chelation (e.g., in the hippocampus) and / or can be lowered or removed by other means. Certain methods and compositions of the invention may be used to bind chelatable copper (e.g., chelatable copper(II)) to reduce copper in the hippocampus while maintaining normal or near-normal copper levels (e.g., within about 70-110% of normal, e.g., 75-105% of normal, 80-100% of normal, or other amounts of copper that are not harmful to the subject).

[0095] The term "pharmaceutical formulation" refers to a preparation that is in such a form that it is capable of effecting the biological activity of the active ingredient contained therein and does not contain additional components that are unacceptably toxic to the subject who is expected to receive the formulation. The pharmaceutical formulation of the present invention useful for lowering hippocampal copper in diabetes comprises or consists essentially of one or more copper antagonists (e.g., one or more copper depleting agents, such as one or more copper chelators or binders (alone or in combination with other therapeutic agents), one or more copper sequestering agents, and / or one or more copper removing agents). As used herein, a pharmaceutical composition is a "pharmaceutical formulation". In some embodiments, the pharmaceutical formulation or composition of the present invention comprises trientine (e.g., triethylenetetramine disuccinate) and a pharmaceutical acceptable carrier. In some embodiments, the pharmaceutical formulation or composition is specifically designed for administration to those with diabetes.

[0096] Therapeutic agents selected from copper antagonists, e.g., copper chelators, copper sequestering agents, copper depleting agents, copper lowering agents, copper removers (alone or in combination with other therapeutic agents), e.g., anti-dementia agents, anti-inflammatory agents, agents for treating cardiovascular disease, agents for treating hypertension, agents for treating kidney disease, agents for treating depression, and agents for treating type 2 diabetes and / or dementia or any of the other disorders disclosed herein, may be administered alone or in combination with one or more additional ingredients and may be formulated into a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, diluents and / or carriers.

[0097] "Pharmaceutically acceptable" refers, for example, to a carrier, diluent, or excipient that is compatible with other components of the formulation and generally safe for administration to a recipient thereof or does not cause undesired adverse physical reactions upon administration. "Pharmaceutically acceptable carrier" as used herein refers to an ingredient in a pharmaceutical formulation other than an active ingredient that can be safely administered to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Pharmaceutically acceptable diluents, carriers, and / or excipients include substances that are useful in the preparation of pharmaceutical compositions and may be co-administered with the compounds described herein while still performing their intended function and that are generally safe, non-toxic, and not biologically or otherwise undesirable. Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for veterinary use as well as human pharmaceutical use. Suitable carriers and / or excipients will be readily apparent to those of skill in the art given the nature of the compounds of the present invention. However, by way of example, diluents, carriers, and / or excipients include solutions, solvents, dispersion media, retardants, polymeric and lipid agents, microspheres, emulsions, etc. By way of further example, suitable liquid carriers, particularly for injectable solutions, include water, saline solutions, aqueous dextrose solutions, etc., for example, isotonic solutions are preferred for intravenous, intrathecal, and intracisternal administration, and vehicles such as liposomes are also suitable for administration of the agents of the invention.

[0098] "Copper chelators" are agents that bind or modify copper (including those that selectively bind and modify copper(I) or copper(II) levels) and are used to lower or normalize blood and / or tissue copper levels and prevent unwanted copper accumulation. Copper chelators include prodrugs thereof. Other agents that normalize copper levels and other agents that selectively bind and modify copper(II) are included in this definition, whether now known or later developed.

[0099] "Copper antagonists" include "copper sequestering agents" and "copper depleting agents" and "copper lowering agents" and are agents that can reduce, bind, and / or inhibit the ability of copper, in any or all of its various forms, e.g., as copper atoms or copper ions (including copper(II) and / or copper(I)), to interact in any chemical or physical reactions that would otherwise be possible in the hippocampus. Copper depleting agents include chelators, agents that reduce total copper, agents that reduce copper levels, agents that reduce intracellular copper (including those described herein). Copper depleting agents also include copper modifying agents, i.e., agents used to reduce hippocampal copper by modifying the content of copper in the body, including intracellular content, or modifying copper availability. It is understood that copper is an essential intracellular nutrient, and thus the present invention includes methods of reducing intracellular copper content while maintaining safe patient copper levels. Copper depleting agents include copper removing agents, i.e., agents that remove copper from the body and / or from cells within. A "copper remover" is a compound that can selectively bind copper and remove copper in complex form from its binding sites in the body, such that the complexed Cu ions are preferentially removed from tissues via plasma into urine or feces and then removed from the body. A "copper remover" compound can be similarly or equally selective for Cu(I), for Cu(II), or for Cu(I) and Cu(II).

[0100] A "compound capable of normalizing copper levels" is a compound that selectively binds to copper (Cu) and removes it from its binding sites in mammalian tissue, such as humans, where the removed Cu is usually in the form of a complex, and through the binding process, the Cu content in the body is restored to a level that is not significantly different from the Cu content in a comparable physiologically normal individual.

[0101] The term "comprising" is synonymous with "including," "containing," or "characterized by," and is not inclusive or limiting and does not exclude additional, unrecited elements or ingredients from the medicament (or step, in the case of a method). The phrase "consisting of" excludes any element, step, or ingredient not specified in the medicament (or step, in the case of a method). The phrase "consisting essentially of" refers to the specified materials and materials that do not materially affect the basic and novel characteristics of the medicament (or step, in the case of a method). The basic and novel characteristics of the present invention are described throughout this specification and include the ability of the compounds, compositions, and methods of the present invention to lower copper levels, lower total copper, lower copper values, lower copper, preferably copper(II), and / or chelate copper, preferably copper(II). Also, the basic and novel features of the present invention include the ability of the compounds, compositions, and methods of the present invention to bring about clinically relevant changes in cognitive impairment in subjects with diabetes or vascular dementia by lowering or normalizing copper, copper levels, and / or total copper, and thus hippocampal copper.In another embodiment of the method of the present invention, the basic and novel features of other compositions and methods of the present invention include the ability to at least partially prevent or reduce the symptoms of cognitive impairment, cognitive decline, and / or dementia, or brain neurodegeneration in subjects with diabetes, such as type 2 diabetes.These concepts include, for example, the ability to reduce or eliminate the harmful effects of excess hippocampal copper.Some symptoms of cognitive impairment, cognitive decline, dementia, and brain neurodegeneration in subjects with diabetes are described herein, but are not limited to these specific symptoms, and include such symptoms known in the art or later identified.

[0102] As used herein, the term "subject" and the like (including "individual" and "patient", all of which may be used interchangeably herein) refers to any mammal, including humans. The preferred mammal herein is a human, including, by way of example, adults, children, including those with diabetes or vascular dementia. In certain embodiments, the subject, individual, or patient is a human. In some embodiments, the subject has hippocampal and / or amygdala atrophy or another hippocampal structural defect. In some embodiments, hippocampal and / or amygdala atrophy or another hippocampal structural defect in a subject with diabetes is visualized using MRI or other imaging devices. In some embodiments, the structural correlates of diabetes-related cognitive impairment in a subject are assessed using brain magnetic resonance imaging (MRI) before and / or during treatment. In some embodiments, in the evaluation of cognitive impairment in patients with diabetes (e.g., patients with type 2 diabetes) for the treatments described herein, MRI is used to test or confirm the presence of normal or abnormal brain structure before and / or during treatment. In some embodiments, for example, white matter hyperintensities are correlated with impaired performance on tests of attention, executive function, information processing speed, and memory in patients with type 2 diabetes, providing a structural basis for treatment with the compounds of the present invention. In some embodiments, MRI is used to test or confirm the presence of white matter hyperintensities before and / or during treatment. In some embodiments, MRI is used to demonstrate or confirm that a subject with diabetes (e.g., type 2 diabetes) has lacunar infarct(s), hippocampal atrophy and / or amygdala atrophy or other hippocampal deficits (including hippocampal atrophy patterns) before treatment with the compounds of the present invention. In some embodiments, MRI is used to evaluate a subject with diabetes (e.g., type 2 diabetes) for hippocampal and / or amygdala atrophy or other hippocampal deficits (including hippocampal atrophy patterns) during treatment with the compounds of the present invention. In some embodiments, MRI is used to assess hippocampal and / or amygdala atrophy in a subject with type 2 diabetes before, during, and / or after treatment according to the methods of the present invention.

[0103] As used herein, "mammal" has its ordinary meaning and includes primates (e.g., humans and non-human primates), laboratory animals (e.g., rodents such as mice and rats), farm animals (such as cows, pigs, mink, sheep, and horses), and domestic animals (such as dogs and cats).

[0104] As used herein, the term "providing to a patient" or "administering to" includes any active or passive manner that ensures the presence in vivo of a compound (e.g., triethylenetetramine disuccinate) to lower or normalize copper, copper value, total copper, etc., including hippocampal copper levels. Preferably, the mode of administration is oral. However, all other modes of administration (particularly parenteral, e.g., intravenous, intramuscular, CNS, etc.) are contemplated. Nasal administration to bypass the blood-brain barrier, administration of the compound of the present invention using a blood-brain barrier penetration enhancer, and cerebrospinal fluid delivery are also contemplated.

[0105] The term "treating cognitive impairment" and the like refers to the prevention, delay, decline, drop, decrease, halt, and / or reversal of cognitive impairment in a subject, or one or more symptoms thereof (e.g., including one or more of the following: general cognition, fluid and crystallized intelligence, information processing speed, psychomotor efficiency, visual and sustained attention, mental flexibility, and / or visual impairment). The term "treating cognitive impairment" and the like may also refer to the prevention, delay, decline, drop, decrease, halt, and / or reversal of cognitive impairment in a subject, or one or more symptoms thereof (e.g., including one or more of the following: memory (such as verbal memory, visual retention, working memory, immediate recall, and / or delayed recall), psychomotor speed and frontal / executive function, processing speed, complex motor function, verbal fluency, and / or attention impairment). In some embodiments, the cognitive impairment interferes with the individual's concentration, memory recall, and / or leads to mental fatigue. The compounds and treatment methods described herein may be used to treat cognitive impairment in diseases, disorders, or conditions characterized by excessive or undesirable copper levels in the hippocampus. The compounds and treatment methods may be used to treat cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration in subjects with diabetes (including, for example, type 2 diabetes). In some embodiments, the subject being treated has type 1 diabetes, and one or more of the cognitive domains negatively affected by cognitive impairment in the subject are general cognition, fluid and crystallized intelligence, information processing speed, psychomotor efficiency, visual and sustained attention, mental flexibility, and / or visual decline. In some embodiments, the subject being treated has type 2 diabetes, and one or more of the cognitive domains negatively affected by cognitive impairment in the subject are memory (verbal memory, visual retention, working memory, immediate recall, delayed recall), psychomotor speed and frontal lobe / executive function, processing speed, complex motor function, verbal fluency, and / or attention. In some embodiments, the cognitive disorder to be treated or prevented is a cognitive disorder that interferes with concentration, memory recall, and / or leads to mental fatigue in individuals with diabetes (e.g., type 2 diabetes).

[0106] "Treating copper overload" refers to the total or partial prevention, delay, lowering, lowering, decreasing, stopping, and / or reversal of pathological excess or undesirable copper in the hippocampus of a subject, and / or treatment of one or more symptoms of excess or undesirable copper. The compounds and treatment methods described herein may be used to treat copper overload. The compounds and treatment methods described herein may be used to remove or reduce undesirable levels or amounts of chelatable copper. The subject is evaluated before and / or during treatment to measure copper in urine (or another source) and / or demonstrate the delay or reversal of hippocampal shrinkage by one or more imaging methods (e.g., by MRI). In some embodiments, excess copper in the subject may be measured by methods such as mass spectrometry of tissue samples, CSF (cerebrospinal fluid) aspirates, and / or urinary copper excretion. In some embodiments, treating copper overload can be measured by a slowing or reversal of hippocampal shrinkage by known imaging methods (e.g., MRI-Magnetic Resonance Imaging) in subjects receiving treatment.

[0107] The term "preventing" refers to prevention or improvement or control in whole or in part. Thus, prevention of cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration refers to prevention or improvement or control in whole or in part of one or more symptoms of one or more of these conditions. The compounds and treatment methods described herein can be used to prevent copper excess, which leads to cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration in diabetes (including, for example, type 2 diabetes).

[0108] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of compound useful for treating hippocampal copper overload. Also, the term "effective amount" or "therapeutically effective amount" is used to refer to the amount of copper antagonist compound for treating and / or preventing cognitive impairment, cognitive decline, and / or dementia, or brain neurodegeneration symptoms in diabetes (including, for example, type 2 diabetes). Triethylenetetramine disuccinate is one such compound. Exemplary effective amounts of this compound are described herein, including doses ranging from about 2400 mg / day to about 3000 mg / day that comprise or essentially consist of a fixed dose of triethylenetetramine disuccinate. In one embodiment, the effective amount of triethylenetetramine disuccinate is at least about 95% pure, at least about 99% pure, or 100% pure. In another embodiment, the effective amount of triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate. In yet another aspect of the method, the effective amount of triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydride.In yet another aspect of the method, the effective amount of triethylenetetramine disuccinate is in the form of a fixed dose tablet or capsule.In one preferred embodiment, the effective fixed dose of triethylenetetramine disuccinate is about 350mg, 400mg, about 500mg, about 600mg, or about 700mg.

[0109] Thus, in one aspect, "effective amount" refers to an amount that is effective to achieve desired therapeutic or preventive results at the required dosage and duration.For example, but not limited to, "effective amount" can refer to the amount of copper antagonists, such as copper sequestering agents or copper depleting agents, that can treat cognitive impairment, cognitive decline, and / or signs and / or symptoms of dementia, or signs of brain neurodegeneration in people with diabetes, by lowering or lowering hippocampal copper or hippocampal free copper (including those disclosed herein, for example, copper chelators such as triethylenetetramine disuccinate).Advantageously, these compounds and methods also reduce copper overload in other regions of the brain.In one embodiment, the effectiveness of the amount is evaluated by determining the subject's response and / or the amount of copper in the subject's urine or plasma after administration of the copper antagonists disclosed herein. Preferably, an effective amount maintains normal copper levels or maintains a subject's copper levels within at least about 70% of normal levels, preferably within at least about 80% of normal levels, at least about 90% of normal levels, or within other levels described herein. In one embodiment, these are urinary copper levels. In other embodiments, serum copper is lowered by no more than about 5-10% (i.e., serum copper is maintained within about 90-95% of normal levels, serving as a safety variable).

[0110] As used herein, "prophylactically effective amount" refers to an amount effective to achieve desired preventive results at the required dosage and duration. Typically, but not necessarily, the prophylactic dose of copper antagonist is used in subjects before or at an earlier stage of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration, so that the prophylactically effective amount may be less than the therapeutically effective amount. Also, the prophylactic dose may function as a maintenance dose once the signs of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration are under control, for example, by one or more initial doses, bolus doses, or loading doses, all as described herein.

[0111] As used herein, the term "treatment" or "treating" of signs and / or symptoms of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration in a mammal means (i) preventing a condition or disease, i.e., avoiding one or more clinical symptoms of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration; (ii) inhibiting cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration, i.e., arresting the onset or progression of one or more clinical symptoms of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration; and / or (iii) alleviating cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration, i.e., causing regression of one or more clinical symptoms (including one or more of the symptoms described herein). Thus, "treatment" (and its grammatical variants such as "treat" or "treating") generally refers to a clinical intervention that seeks to alter the natural course of the individual, tissue, or cell being treated, and can be performed either for prophylaxis or in the course of clinical pathology. The term does not necessarily mean that the subject is treated until complete recovery. Thus, "treatment" includes lowering, lowering, reducing, or improving the symptoms or severity of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration, or preventing or otherwise reducing the risk of developing cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration. "Treatment" may also include maintaining or promoting a state of complete or partial remission of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration. The copper antagonist compounds described herein (including, for example, triethylenetetramine disuccinate) are used for treatment.

[0112] As used herein, "associated with" merely means that both situations exist, and should not be construed as meaning that one is necessarily causally related to the other.

[0113] Structural correlates of diabetes-related cognitive impairment can be assessed using brain magnetic resonance imaging (MRI) before and / or during treatment, as described and claimed herein. See Jongen C, Biessels GJ: Structural brain imaging in diabetes: a methodological perspective. Eur J Pharmacol 2008;585:208-218.

[0114] In some embodiments, as described, in the assessment of cognitive impairment in patients with diabetes (e.g., type 2 diabetes) for treatment (or during treatment) as described herein, magnetic resonance imaging (MRI) is used to test or confirm the presence of normal or abnormal brain structure before and / or during treatment. In some embodiments, in patients with type 2 diabetes, for example, white matter hyperintensities are correlated with impaired performance on tests of attention, executive function, information processing speed, and memory, providing a structural basis for treatment with the compounds of the present invention. In some embodiments, as described, MRI is used to demonstrate or confirm that subjects with type 2 diabetes have atrophy of the hippocampus and / or amygdala before and / or during treatment with the compounds of the present invention. In some embodiments, MRI is used to assess the normality and / or signs of atrophy or shrinkage of the hippocampus and / or amygdala in subjects with diabetes before and / or during treatment with the compounds of the present invention. Also, the middle temporal gyrus and / or entorhinal cortex may be evaluated for abnormalities before treatment and / or repair during treatment. In some embodiments, the subject has type 2 diabetes. The hippocampus and amygdala are responsible for functions such as memory and behavior, and interestingly, have also been found to be atrophied in Alzheimer's patients.

[0115] The present invention provides a method for treating or preventing cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration in a subject with diabetes, comprising administering to the subject a pharmaceutical composition comprising a compound capable of lowering, lowering, or normalizing hippocampal copper levels or amounts. In some embodiments, the compound is capable of treating copper overload. In some embodiments, the compound is a copper antagonist. In some embodiments, the copper antagonist is a copper chelator or other copper binding agent or a copper removing agent or a sequestering agent. In some embodiments, the copper chelator or other copper binding agent is trientine. In some embodiments, the trientine is triethylenetetramine disuccinate. In other embodiments, the trientine is triethylenetetramine dihydrochloride or tetrahydrochloride. In other embodiments, the compound is another compound effective to reduce copper, lower total copper, or lower copper content, and thus hippocampal copper, in the subject, as described herein. In other embodiments, the compound is another compound effective to reduce copper, lower total copper, or lower copper content in a subject, and thus, hippocampal copper is copper currently known in the art or later identified.

[0116] The present invention also provides methods for treating or preventing cognitive and / or memory impairment in a subject with diabetes. In some embodiments, the subject has type 2 diabetes. In other embodiments, the subject has type 1 diabetes. In some embodiments, the subject has type 3 diabetes. In some embodiments, the subject has type 4 diabetes.

[0117] In some embodiments, the invention provides methods of treating a subject with diabetes (e.g., type 1 diabetes) and a decline in one or more of overall cognition, fluid and crystallized intelligence, speed of information processing, psychomotor efficiency, visual and sustained attention, mental flexibility, and / or vision with a copper antagonist by administering one or more compounds or compositions of the invention to improve one or more of overall cognition, fluid and crystallized intelligence, speed of information processing, psychomotor efficiency, visual and sustained attention, mental flexibility, and / or vision in the subject.

[0118] In some embodiments, the present invention provides methods of treating a subject with diabetes (e.g., type 2 diabetes) and one or more negatively affected cognitive domains, including memory (verbal memory, visual retention, working memory, and / or immediate and / or delayed recall), psychomotor speed and frontal / executive function, processing speed, complex motor function, verbal fluency, and / or attention, by administering one or more compounds or compositions of the present invention to improve one or more of these or other negatively affected cognitive domains in the subject. In some embodiments, the cognitive impairment to be treated or prevented is a cognitive impairment that interferes with concentration, memory recall, and / or leads to mental fatigue in individuals with diabetes (e.g., type 2 diabetes).

[0119] In other embodiments, the present invention relates to a method for treating or preventing cognitive impairment or decline in a subject with vascular dementia, sometimes referred to as vascular cognitive impairment or VCI, comprising administering to the subject a pharmaceutical composition comprising a copper antagonist, including a compound capable of reducing, lowering, and / or normalizing copper metabolism. In some embodiments, the subject may or may not have diabetes. In some embodiments, the copper antagonist compound is a copper chelator or other copper binding agent. In some embodiments, subjects with vascular dementia or VCI have problems with reasoning, planning, judgment, memory, and other thought processes, and the compounds, compositions, and methods of the present invention reduce and alleviate these problems in whole or in part.

[0120] In some embodiments of the method of the present invention, the compound capable of normalizing copper metabolism can reduce or alleviate the elevated copper level in a subject and increase the decreased copper level.In some embodiments, the compound capable of lowering the elevated copper level in a subject and increase the decreased copper level is trientine.In some embodiments, trientine is triethylenetetramine disuccinate.

[0121] In some embodiments of the methods of the present invention, the copper antagonist is 2+ In some embodiments, the copper binding compound is a copper chelator. In some embodiments, the copper chelator is a copper binding compound that binds to copper. 2+ In some embodiments of the methods of the present invention, the copper antagonist chelates copper. 1+ In some embodiments, the copper binding compound is a copper chelator. In some embodiments, the copper chelator is a copper binding compound that binds to copper. 1+ In some embodiments, the copper chelator chelates copper. 1+ and copper 2+ In some embodiments, the agent chelates Cu. 1+ In some embodiments, the agent preferentially binds to Cu. 2+In some embodiments, Cu 2+ In some embodiments, the agent that preferentially binds to Cu is triethylenetetramine disuccinate. 1+ and Cu 2+ In one embodiment, Cu 1+ and Cu 2+ An agent that preferentially binds to is a penicillamine copper chelator, preferably D-penicillamine.

[0122] In some embodiments, the pharmaceutical composition used in the methods of the present invention comprises a therapeutically effective amount of triethylenetetramine and a pharma- ceutically acceptable carrier, glidant, diluent, or excipient. In some embodiments, the triethylenetetramine is in the form of a pharma- ceutically acceptable salt.

[0123] In one embodiment, the copper depleting agent or copper antagonist is an agent effective for lowering copper content or total copper and hippocampal copper in a subject. In another embodiment, the agent administered to a subject with diabetes and / or vascular dementia comprises, consists essentially of, or consists of an agent that binds or chelates copper(II). In another embodiment, the agent comprises, consists essentially of, or consists of an agent that binds or chelates copper(I). In another embodiment, the agent comprises, consists essentially of, or consists of an agent that binds or chelates both copper(I) and copper(II).

[0124] In one embodiment, copper antagonists or agents effective in lowering copper content or otherwise removing excess hippocampal copper in a subject include D-penicillamine; N-acetylpenicillamine; triethylenetetramine (also known as TETA, TECZA, triene, triene, and trientine) and pharma- ceutically acceptable salts thereof; trithiomolybdate, tetrathiomolybdate, ammonium tetrathiomolybdate, choline tetrathiomolybdate; bis-choline tetrathiomolybdate (thiomolybdate USAN, trade name Decuprate), 2,2,2 tetramine tetrahydrochloride; 2,3,2 tetramine tetrahydrochloride; Ethylenediaminetetraacetate (EDTA, a non-preferential, non-specific metal binder that is dosed with caution to avoid toxicity); diethylenetriaminetetraacetic acid (DPTA, a non-preferential, non-specific metal binder that is dosed with caution to avoid toxicity due to chelation of essential metals (such as Zn and Mn); 5,7,7'12,14,14'hexakismethyl-1,4,8,11 tetraazacyclothretradecane; 1,4,8,11 tetraazacyclothretradecane, e.g., cyclam S, cyclamus, and copper-chelating cyclam derivatives, e.g., Bn-cyclam-EtOH, oxo-cyclam-EtOH and oxo-Bn-cyclam-EtOH, (HOCH 2 CH 2 CH 2 ) 2 (PhCH 2 ) 2 Cyclam, and [ka] ;1,4,8,11-Tetraazabicyclotetradecane-1,4,8,11-tetraacetic acid;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;Melatonin;Cyclic 3-hydroxymelatonin (3OHM);N(1)-Acetyl-N(2)-formyl-5-methoxykynuramine (AFMK);N(1)-Acetyl-5-methoxykynuramine (AMK);N,N'-Diethyldithiocarbamate;Bathocuproine The composition may comprise, consist essentially of, or consist of an agent selected from the group consisting of disulfonic acids; bathocuprine disulfonate; trimetazidine; triethylenetetramine tetrahydrochloride; 2,3,2-tetraamine; 1,10-orthophenanthroline; 3,4-dihydroxybenzoic acid; 2,2'-bicinchoninic acid; diamsar; 3,4',5, trihydroxystilbene (resveratrol); mercaptodextran; disulfiram (Antabuse); sarcofagin; DiAmSar; diethylenetriaminepentaacetic acid; and diethylenetriaminepentaacetic acid calcium trisodium; neocuproine; bathocuproine; and carnosine.

[0125] In one embodiment, the agent reduces hippocampal copper in the subject. In some embodiments, the agent lowers total copper in the subject. In some embodiments, the copper antagonist or copper-lowering agent maintains total copper in the subject within the range of normal human serum or plasma of about 0.8-1.2 milligrams / L, or about 10-25 micromoles / L. In some embodiments, the copper-lowering agent maintains total copper in the subject within at least about 70% (e.g., at least about 75%) of the normal range of about 0.8-1.2 milligrams / L, or about 10-25 micromoles / L. In some embodiments, the copper-lowering agent maintains total copper in the subject within about 75% to about 85%, or about 85% to about 95% of the normal range of copper in human plasma or serum. In some embodiments, one aspect of a subject's copper status provided by a copper antagonist or other copper lowering agent or agent for addressing elevated hippocampal or brain copper according to the methods of the invention is determined by assessing the level or amount of copper in the subject's urine.

[0126] In some embodiments, the compound administered to the subject in the practice of any method of the invention is triethylenetetramine. In some embodiments, triethylenetetramine is the hydrochloride salt of triethylenetetramine. In one embodiment, triethylenetetramine hydrochloride is triethylenetetramine dihydrochloride. In another embodiment, triethylenetetramine hydrochloride is triethylenetetramine tetrahydrochloride. In another embodiment, triethylenetetramine is the succinate salt of triethylenetetramine. In one embodiment, triethylenetetramine succinate salt is triethylenetetramine disuccinate. In one aspect of the invention, the method uses a crystalline form of triethylenetetramine disuccinate or the hydrochloride salt of triethylenetetramine. In another aspect of the invention, the method uses triethylenetetramine disuccinate anhydrous or the hydrochloride salt of triethylenetetramine anhydrous.

[0127] In one embodiment of the invention, the method employs a pharmaceutical composition comprising substantially pure triethylenetetramine disuccinate. In another embodiment, the method employs a pharmaceutical composition comprising substantially pure triethylenetetramine disuccinate and a pharma- ceutically acceptable excipient. In another embodiment, the method employs a pharmaceutical composition comprising substantially pure triethylenetetramine dihydrochloride or tetrahydrochloride and a pharma- ceutically acceptable excipient. A preferred pharmaceutical composition for use in the method of the invention comprises, consists essentially of, or consists of substantially pure triethylenetetramine disuccinate. Another preferred composition comprises, consists essentially of, or consists of substantially pure triethylenetetramine disuccinate anhydride. Another preferred composition is a composition comprising, consists essentially of, or consists of substantially pure triethylenetetramine disuccinate crystals having alternating layers of triethylenetetramine and succinate molecules.

[0128] In certain embodiments, the triethylenetetramine succinate is a triethylenetetramine disuccinate polymorph. Triethylenetetramine disuccinate polymorphs have been described in the art. In certain embodiments, the triethylenetetramine hydrochloride is a triethylenetetramine hydrochloride polymorph.

[0129] In some embodiments, the present invention includes a method for treating cognitive impairment in subjects with diabetes and excess hippocampal copper, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of trientine, the succinic acid addition salt of triethylenetetramine, the hydrochloric acid addition salt of triethylenetetramine, and a pharma- ceutically acceptable salt of D-penicillamine, N-acetylpenicillamine, tetrathiomolybdate, ammonium tetrathiomolybdate, and choline tetrathiomolybdate. In some embodiments, the compounds are used to treat and / or prevent cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration in these subjects, including subjects with type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, and / or vascular dementia.

[0130] In some embodiments of the methods of the present invention, the subject exhibits signs of brain degeneration prior to treatment. In other embodiments, the subject has or is at risk of having cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration.

[0131] In some embodiments, subjects with diabetes and / or vascular dementia have impaired spatial memory or the ability to remember directions, location, and orientation.

[0132] In some embodiments of the methods of the present invention, the methods further comprise administering one or more additional therapeutic agents selected from an anti-inflammatory agent, an agent for treating cardiovascular disease, an agent for treating hypertension, an agent for treating kidney disease, an agent for treating depression, and an agent for treating type 2 diabetes and / or dementia.

[0133] In some embodiments, the one or more additional therapeutic agents for treating type 2 diabetes are selected from the group consisting of alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 receptor agonists, meglitinides, sodium glucose transporter (SGLT) 2 inhibitors, sulfonylureas, and thiazolidinediones.

[0134] In some embodiments, the one or more additional therapeutic agents for treating dementia are selected from the group consisting of cholinesterase inhibitors, antibodies targeting amyloid beta protein (a biomarker for Alzheimer's disease and other dementias), and N-methyl-D-aspartate (NMDA) receptor antagonists. In some embodiments, the cholinesterase inhibitor is donepezil (Aricept), galantamine (Razadyne, Razadyne ER, Reminyl), or Rivastigmine (Exelon). In some embodiments, the antibody targeting amyloid beta protein is Aducanumab-avwa (Aduhelm). In some embodiments, the NMDA receptor antagonist is memantine (Axura, Ebixa, Namenda, etc.).

[0135] In some embodiments, the subject is a human.

[0136] In some embodiments, the pharmaceutical composition is administered orally in the form of a capsule or tablet.

[0137] In some embodiments, the compound is triethylenetetramine dihydrochloride and is administered in an amount of about 1200 mg daily. In some embodiments, 1200 mg of triethylenetetramine dihydrochloride is administered in 600 mg divided doses BID, in 400 mg divided doses TID, or in 300 mg divided doses QID.

[0138] In some embodiments, the compound is triethylenetetramine disuccinate and is administered in a dose ranging from about 2400 mg / day to about 3000 mg / day or more. In some embodiments, the compound is triethylenetetramine disuccinate and is administered in an amount of about 2800 mg / day. Other useful doses of triethylenetetramine disuccinate are administered, such as about 1050 mg / day to about 2300 mg / day, about 1400 mg / day to about 3500 mg / day, about 2400 mg / day to about 3200 mg / day, and about 2800 mg / day to about 5600 mg / day. In some embodiments, these daily amounts of triethylenetetramine disuccinate are administered in divided doses.

[0139] In another aspect of the invention, the methods of the invention maintain copper levels at about 70% to about 100% of normal levels in a subject, thereby lowering copper values ​​and / or inducing lowering or lowering of copper levels in a mammalian patient. In some embodiments, the methods of the invention maintain copper levels at about 70% to about 110% of normal levels in a subject. Urinary copper excretion values ​​may be up to about 300-35% of normal levels immediately after treatment begins, and these and these values ​​typically decline to about 200-250% of normal levels after 4-12 months.

[0140] In certain embodiments, triethylenetetramine disuccinate is administered after an initial dose (or loading dose), where the loading dose is about or at least 1.5 times, about or at least 2 times, about or at least 2.5 times, or about or at least 3 times the maintenance dose. The maintenance dose may be, for example, about 350 mg, 400 mg, about 500 mg, about 584 mg, about 600 mg, and / or about 700 or 701 mg, 1 to 4 times daily. In one embodiment, the loading dose may be administered 1, 2, 3, 4, or 5 times prior to the initial maintenance dose, and 1, 2, 3, or 4 times daily.

[0141] Thus, for example, in one embodiment, for a loading dose regimen of 2337 mg triethylenetetramine disuccinate daily, triethylenetetramine disuccinate is administered at a daily loading dose (which can be provided in one or several doses throughout the day) of at least about 3505 mg (1.5 times), at least about 4674 mg (2 times), at least about 5842 mg (2.5 times), or at least about 7001 mg (3 times). In one embodiment, the loading dose of triethylenetetramine disuccinate is administered in two doses per day, for one, two, three, four, or five days or more, as needed. Thus, the loading dose of other triethylenetetramine disuccinate is calculated based on the maintenance dose of triethylenetetramine disuccinate administered daily or at other frequencies (such as, for example, 2804 or other maintenance dose administered daily).

[0142] In one embodiment, triethylenetetramine disuccinate or other copper antagonists as described herein are administered twice a day (BID) to provide desired daily dosage.In another embodiment, triethylenetetramine disuccinate or other copper antagonists as described herein are administered three times a day (TID) to provide desired daily dosage.In yet another embodiment, dose is administered four times a day (QID) to provide desired daily dosage.

[0143] Doses of triethylenetetramine disuccinate described herein and in the claims are used to selectively bind and alter copper(II) levels, prevent or reduce or normalize blood and / or tissue copper levels, and prevent and / or reduce unwanted copper accumulation in the hippocampus, and are administered to subjects with elevated hippocampal copper (or at risk for elevated hippocampal copper and signs of cognitive impairment, cognitive decline, and / or dementia, or brain neurodegeneration).

[0144] Triethylenetetramine disuccinate includes prodrugs thereof with dosages modified to account for the molecular weight of the "pro-" portion of the triethylenetetramine disuccinate prodrug.

[0145] The dose of triethylenetetramine disuccinate or another copper antagonist may be administered alone or in combination with one or more additional components, and may be formulated into a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, diluents, and / or carriers. In some embodiments, the present invention provides a combination product comprising (a) a dose of triethylenetetramine disuccinate or another copper antagonist, and (b) a therapeutic agent comprising one or more anti-dementia and / or anti-diabetic agents, where components (a) and (b) are adapted for simultaneous or sequential administration. In some embodiments, component (b) is an anti-inflammatory agent, an agent for treating cardiovascular disease, an agent for treating hypertension, an agent for treating kidney disease, an agent for treating depression, and / or an agent for treating type 2 diabetes. In certain embodiments of the present invention, the combination product according to the present invention is used in such a manner that at least one of the components is administered while the other components still exert an effect on the subject being treated. The doses of triethylenetetramine disuccinate or other copper antagonist and component (b) therapeutic agent may be administered separately in the same or one or more different containers, or may be mixed and administered together in any combination. Preferably, both or all three of the triethylenetetramine disuccinate (or other copper antagonist) and antidiabetic drug and / or therapeutic agent are combined in a capsule for oral administration.

[0146] In another embodiment, the copper level, preferably copper 2+Compounds capable of copper chelation or copper normalization by lowering or increasing the level or amount of (e.g., triethylenetetramine disuccinate) are used in combination with agents for the treatment of diabetes. Agents for the treatment of diabetes according to the invention described herein and claimed include alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 receptor agonists, meglitinides, sodium glucose transporter (SGLT) 2 inhibitors, sulfonylureas, and thiazolidinediones. Alpha-glucosidase inhibitors for use in the invention described herein and claimed include, for example, acarbose (Precose) and miglitol (Glyset). The most common biguanide is metformin (Glucophage, metformin hydrochloride ER, Glumetza, Riomet, Fortamet). Metformin for use in the present invention as described herein and claimed may also be combined with other drugs for type 2 diabetes and is an ingredient in the following medicines: metformin-alogliptin (Kazano); metformin-canagliflozin (Invokamet); metformin-dapagliflozin (XigduoXR); metformin-empagliflozin (Synjardy); metformin-glipizide; metformin-glyburide (Glucovance); metformin-linagliptin (Jentadueto); metformin-pioglitazone (Actoplus); metformin-repaglinide (PrandiMet); metformin-rosiglitazone (Avandamet); metformin-saxagliptin (KombiglyzeXR); and metformin-sitagliptin (Janumet). Bromocriptine (Cycloset) is a dopamine agonist that may be used in the present invention as described and claimed herein.Dipeptidyl peptidase-4 (DPP-4) inhibitors for use in the present invention as described and claimed herein include: alogliptin (Nesina); alogliptin-metformin (Kazano); alogliptin-pioglitazone (Oseni); linagliptin (Tradjenta); linagliptin-empagliflozin (Glyxambi); linagliptin-metformin (Jentadueto); saxagliptin (Onglyza); saxagliptin-metformin (Kombiglyze XR); sitagliptin (Januvia); sitagliptin-metformin (Janumet and Janumet XR); and sitagliptin and simvastatin (Juvisync). Glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists) for use in the present invention as described and claimed herein include: albiglutide (Tanzeum); dulaglutide (Trulicity); exenatide (Byetta); exenatide extended release (Bydureon); liraglutide (Victoza); and semaglutide (Ozempic). Meglitinides for use in the present invention as described and claimed herein include: nateglinide (Starlix); repaglinide (Prandin); and repaglinide-metformin (Prandimet). Sodium-glucose transporter (SGLT) 2 inhibitors for use in the present invention as described and claimed herein include: dapagliflozin (Farxiga); dapagliflozin-metformin (XigduoXR); canagliflozin (Invokana); canagliflozin-metformin (Invokamet); empagliflozin (Jardiance); empagliflozin-linagliptin (Glyxambi); empagliflozin-metformin (Synjardy); and ertugliflozin (Steglatro).Sulfonylureas for use in the present invention as described and claimed herein include: glimepiride (Amaryl); glimepiride-pioglitazone (Duetact); glimepiride-rosiglitazone (Avandaryl); gliclazide; glipizide (Glucotrol); glipizide-metformin (Metagrip); glyburide (DiaBeta, Glynase, Micronase); glyburide-metformin (Glucovance); chlorpropamide (Diabinese); tolazamide (Tolinase); and tolbutamide (Orinase, Tol-Tab). Thiazolidinediones for use in the present invention as described and claimed herein include: rosiglitazone (Avandia); rosiglitazone-glimepiride (Avandaryl); rosiglitazone-metformin (Amaryl M); pioglitazone (Actos); pioglitazone-alogliptin (Oseni); pioglitazone-glimepiride (Duetact); and pioglitazone-metformin (Actoplus Met, Actoplus Met XR).

[0147] Such combination products may be prepared according to the methods and principles provided herein as well as those known in the art. Also provided are combination products for use in the methods described herein.

[0148] When administered individually or together, the formulations of copper antagonists, copper chelators, or other copper lowering agents (e.g., triethylenetetramine disuccinate formulations) can be prepared to provide rapid or slow release; immediate, delayed, timed, or sustained release; or combinations thereof. The formulations can be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, electuaries, drops (including, but not limited to, eye drops), tablets, granules, powders, lozenges, sachets, capsules, gels, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols. For example, gastroretentive or mucoadhesive formulations of triethylenetetramine disuccinate can enhance or prolong the absorption of this therapeutic product in the gastrointestinal tract. The delayed release form of triethylenetetramine disuccinate serves to avoid metabolism, prolong and increase absorption, and increase bioavailability by releasing the drug after it has passed through the stomach.A variety of different means are available to achieve these modified release formulations.Such technologies are well known to those skilled in the art, and the particular technology and excipients are selected to address the problems or challenges posed by the ADME profile of the product in question.

[0149] Mucoadhesive formulations contain certain polymers that adhere to the epithelial lining of hydrated sites. Thus, for example, a drug released into the duodenum after passing through the stomach will adhere to the walls of the digestive tract, resulting in prolonged and preferential release and absorption of the drug from this site. Buccal, corneal, respiratory, and vaginal tissues are also targets for such formulations, as they are covered with mucosal tissue. The mucoadhesiveness of most polymers increases with molecular weight, so, for example, MW in the range of 200,000 to 700,000 has been found to correlate with improved membrane adhesion for polyoxyethylene polymers and copolymers. Viscosity, pore size, and degree of crosslinking are other factors considered in the selection of mucoadhesive polymers. Hydrogen bonding, flexibility, degree of hydration, and swelling are also important factors in drug delivery from mucoadhesive polymers. In addition to polyoxyethylene / polyvinyl alcohol, materials composed of polymers of acrylic and methacrylic acid esters, as well as hydroxylated methacrylic acid polymers, are useful for this purpose. Chitosan, cyanoacrylate, hyaluronic acid, hydroxypropylcellulose, gellan, polycarbopole, and sodium carboxymethylcellulose are other related polymers used in mucoadhesive formulations. Nasal mucoadhesive formulations are developed with an eye on the specific properties of such tissues. Nasal delivery systems include copolymers of methyl vinyl ether, (hydroxypropyl)methylcellulose (HPMC), sodium carboxymethylcellulose, carbopol-934P, and Eudragit RL-10. Mucin, gelatin, polycarbophil, and poloxamer are examples of polymers used for vaginal or rectal mucoadhesive formulations. Oral delivery systems for GI mucoadhesive systems are represented by chitosan, polyacrylic acid, alginate, polymethacrylic acid, and sodium carboxymethylcellulose. Fixed dose mucoadhesive triethylenetetramine disuccinate formulations can be prepared using such compounds.

[0150] Gastroretentive formulations are generally designed for drugs that have an optimal window of absorption in the stomach and proximal intestine. Hydrodynamically balanced systems, floating microspheres, gas-generating tablets, formulations that swell to prevent passage from the stomach, and formulations that adhere to the stomach wall are examples of such formulations. A "plug" system that expands to a size that does not easily pass the pyloric sphincter is an example of a gastroretentive formulation. Low density (buoyant) or gas-generating (carbon dioxide) formulations are retained for extended periods of time; such techniques may be used in combination to optimize such performance. Mucoadhesive polymers are also often used to design such effects into the formulation. Sodium alginate can be combined with sodium carbonate or sodium bicarbonate to achieve a "rafting" effect, resulting in the formulation being retained in the stomach based on buoyancy in gastric juices. Fixed dose gastroretentive triethylenetetramine disuccinate formulations can be prepared using these methods and compounds.

[0151] In some embodiments, the copper antagonist or copper lowering agent is substantially pure (including at least about 90% pure, at least about 95% pure, and 100% pure). In some embodiments, the copper antagonist or copper lowering agent is triethylenetetramine. In some embodiments, the triethylenetetramine is triethylenetetramine disuccinate. In some embodiments, the triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydride. In some embodiments, the triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate or triethylenetetramine disuccinate anhydride. In some embodiments of the present invention, the copper antagonist or copper lowering agent is a polymorph of triethylenetetramine disuccinate. Triethylenetetramine disuccinate polymorphs are described, for example, in U.S. Pat. No. 8,067,641. In one embodiment, the copper antagonist or copper lowering agent comprises a polymorph of triethylenetetramine disuccinate, wherein the polymorph is a crystal having a structure defined by the coordinates of Table 3B found in U.S. Patent No. 8,067,641. In another embodiment of the invention, the copper antagonist or copper lowering agent comprises a polymorph of triethylenetetramine disuccinate, wherein the polymorph is a crystal having a structure defined by the coordinates of Table 3C found in U.S. Patent No. 8,067,641. In another embodiment of the invention, the triethylenetetramine disuccinate consists essentially of a triethylenetetramine disuccinate polymorph having a crystal having a structure defined by the coordinates of Table 3B in U.S. Patent No. 8,067,641, or consists essentially of a crystalline triethylenetetramine disuccinate polymorph having a structure defined by the coordinates of Table 3C in U.S. Patent No. 8,067,641.

[0152] Fixed dose and daily or other cycles of administration The effective fixed dose of triethylenetetramine disuccinate is about 400mg, about 500mg, about 600mg, or about 700mg. A fixed dose of 350mg is also provided. For example, the fixed dose is used to administer a dose of triethylenetetramine disuccinate ranging from about 2400mg / day to about 3000mg / day, or other time period. In one embodiment, the effective amount of triethylenetetramine disuccinate is at least about 95% pure, at least about 99% pure, or 100% pure. In another embodiment, the effective amount of triethylenetetramine disuccinate is a crystalline form of triethylenetetramine disuccinate. In yet another embodiment of the method, the effective amount of triethylenetetramine disuccinate is triethylenetetramine disuccinate anhydride. In yet another embodiment of the method, the effective amount of triethylenetetramine disuccinate is in the form of a fixed dose tablet or capsule. The total dosage can be administered in single or divided doses (e.g., BID, TID) and preferably maintains normal urinary and / or plasma copper levels in the subject, or levels not below about 70%-75% of normal levels. In one preferred embodiment, a fixed dose is administered BID.

[0153] manufacturing Triethylenetetramine disuccinate suitable for use in the present invention can be obtained from known sources or synthesized using methods known in the art.Preparation methods are described, for example, in U.S. Patent No. 9,556,123, which describes the synthesis of triethylenetetramine and intermediates useful in its production.U.S. Patent No. 8,067,641 describes the synthesis of substantially pure triethylenetetramine disuccinate, substantially pure triethylenetetramine disuccinate anhydride, and triethylenetetramine disuccinate polymorphs.

[0154] Pharmaceutical Preparations Also provided is a pharmaceutical preparation comprising a fixed dose of a copper antagonist (e.g., triethylenetetramine, including triethylenetetramine disuccinate) present in a pharmaceutically acceptable vehicle. The term "pharmaceutically acceptable" has the above meaning and includes vehicles approved by federal or state regulatory agencies for use in mammals, such as humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compound of the present invention is formulated for administration to a mammal.

[0155] In one aspect, the disclosure provides a pharmaceutical preparation in which a copper antagonist (e.g., triethylenetetramine disuccinate), alone or with other active ingredients, is prepared by combining the copper antagonist (or copper antagonists) with one or more pharma- ceutically acceptable diluents, carriers, adjuvants, etc., in a manner known to those skilled in the art of pharmaceutical formulation. Dosage forms can be prepared by combining with one or more pharma- ceutically acceptable diluents, carriers, adjuvants, etc., in a manner known to those skilled in the art of pharmaceutical formulation.

[0156] The choice of excipient will be determined in part by the active ingredient, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

[0157] Dosage forms useful herein include any appropriate dosage form known in the art to be suitable for pharmaceutical formulations of compounds suitable for administration to mammals, particularly humans, particularly dosage forms suitable for stabilization (although not solely) of solutions, tablets, or capsules containing a therapeutic compound for administration to humans.

[0158] The composition may take the form of any standard known dosage form, such as those mentioned above, e.g., tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, injectable solutions, transdermal delivery devices (e.g., transdermal patches), or any other suitable composition. Those skilled in the art to which the present invention pertains will recognize the most appropriate dosage form without any undue experimentation, taking into consideration the nature of the condition to be treated and the active agent to be used. Various doses and dosage ranges, including doses and dosage ranges of triethylenetetramine disuccinate, are described herein. It should be recognized that one or more of the other active agents (e.g., anti-inflammatory agents, etc. and others described) may be formulated into a single composition with the copper antagonist dose. In certain embodiments, preferred dosage forms include injectable solutions, topical formulations in transdermal patches, and oral formulations. The dosage forms of the present invention include any suitable dosage form now known in the art or later discovered to be suitable for pharmaceutical formulations of compounds suitable for administration to humans.

[0159] An example is an oral delivery form such as a tablet, capsule, or lozenge, or in the case of an oral dosage form, any liquid form that can protect the compound from degradation before it takes effect, for example, in the gastrointestinal tract. Particular formulations for use in the present invention are solid forms, particularly tablets or capsules for oral administration.

[0160] Slow or controlled release (e.g., triethylenetetramine disuccinate) copper antagonist preparations in tablets or capsules are preferred. Oral, parenteral, etc., as well as intranasal administration to bypass the blood-brain barrier, are one method of administration.

[0161] In addition to standard diluents, carriers, and / or excipients, the composition according to the present invention may be formulated with or in such a way as to enhance activity or bioavailability, help protect integrity, increase its half-life or shelf life, allow slow release upon administration to a subject, or provide other desirable advantages. For example, slow release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or hydrogels to allow the product to be released from the matrix over time. As further examples, the composition may also include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, coating agents, and buffers. Those skilled in the art of the present invention will easily identify additional additives that may be desirable for a particular purpose.

[0162] The dose of the copper antagonist of the present invention can also be administered by sustained release system.Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles (e.g., films or microcapsules).Sustained release matrices include polylactides (U.S. Pat. No. 3,773,919; European Patent No. 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid (European Patent No. 133,988).Sustained release compositions also include compounds that are enclosed (e.g., encapsulated) in liposomes. Liposomes containing copper chelating agents (alone or with antiviral and / or anti-inflammatory agents) can be prepared by known methods, including, for example, those described in German Patent No. 3,218,121; European Patent No. 52,322; European Patent No. 36,676; European Patent No. 88,046; European Patent No. 143,949; European Patent No. 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and European Patent No. 102,324. Typically, liposomes may be used to encapsulate triethylenetetramine disuccinate and are small (about 200 to 800 angstroms) unilamellar type with lipid content exceeding about 30 mole percent cholesterol, with the ratio selected and adjusted for the most effective treatment. For example, slow release delivery using PGLA nanoparticles or microparticles, or an in situ ion-activated gelation system may also be used.

[0163] In one such example, the desired salt form can be formulated using a gastric retentive dosage form (GRDF). Such delivery forms are formulated with the intention of extending gastric retention time and therefore enhancing absorption. Such strategies can use, for example: 1) transit retardants, 2) large single unit dosage forms, 3) bioadhesive drug delivery systems, 4) heavy pellets, 5) buoyant forms. Polymers such as carbopol, chitosan, sodium alginate, HPMC, polyacrylic acid, polyethylene glycol, and modified forms of these polymers are variously used to achieve gastric retention, among other examples.

[0164] In the second example of the dosage form modified for non-immediate release, the product is formulated to delay the release of drug until the dosage form leaves the stomach.In the delayed release form, the release profile is similar or equivalent to that of the immediate release form, but the actual release of drug is delayed, for example, by enteric coating, so that the active ingredient is not released from the granules of the dosage form until it has completely passed through the stomach.The enteric coating as an example of this strategy is achieved, for example, by using (meth)acrylic polymer that does not dissolve in aqueous medium until the pH is above 5.5, resulting in a dosage form that passes through the stomach without releasing the copper antagonist active ingredient.

[0165] A sustained release dosage form differs from delayed release in that the release profile of the drug is extended beyond that of an immediate release product. Mechanisms of sustained release include delayed dissolution, diffusion, delivery from the intact dosage form by osmotic pressure, maintaining hydrological or hydrodynamic balance, and ion exchange. The traditional means of obtaining sustained release is to formulate in a matrix of non-ionic cellulose ethers (such as HPMC, see US8865778B2) in the presence of a selected amount of a non-crosslinked swelling agent (such as carboxymethyl starch or sodium starch glycolate). Other approaches to achieve the same result are known. For example, the core of a drug delivery formulation containing an osmotic agent and a water-swellable polymer is easily used as a driving force to deliver the drug in a controlled and extended manner.

[0166] Therapeutic formulations for use in preparing the methods and compositions of the present invention may be prepared by any of the methods well known in the art of pharmacy. For example, Gilman et al. (eds.) GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS (8th ed.) Pergamon Press (1990); and Remington, THE SCIENCE OF PRACTICE AND PHARMACY, 20th Edition. (2001) Mack Publishing Co., Easton, Pa.; Avis et al. al. (eds.) (1993) PHARMACEUTICAL DOSAGE FORMS: PARENTERAL MEDICATIONS Dekker, NY; Lieberman et al. (eds.) (1990) PHARMACEUTICAL DOSAGE FORMS: TABLETS Dekker, NY; and Lieberman et al. (eds.) (1990) PHARMACEUTICAL DOSAGE FORMS: DISPERSE SYSTEMS See Dekker, NY. Compositions may also be formulated according to standard techniques which may be found in standard references such as, for example, Gennaro AR: Remington: The Science and Practice of Pharmacy, 20.sup.th ed., Lippincott, Williams & Wilkins, 2000.

[0167] Certain formulations of the invention are in the form of intranasal administration, such as nanoemulsions. Other formulations of the invention are in the form of transdermal patches.

[0168] Manufactured products / kits The present invention also provides an article of manufacture, or "kit," containing materials useful for treating or preventing cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration in a subject with diabetes (e.g., type 2 diabetes) and / or vascular dementia (e.g., VCI), comprising: (a) a pharmaceutical composition comprising a copper antagonist compound capable of lowering, lowering, and / or normalizing hippocampal copper levels or amounts; and (b) instructions for use in therapeutically treating or preventing cognitive impairment, cognitive decline, and / or dementia, or symptoms of brain neurodegeneration in a subject with diabetes and / or vascular dementia (e.g., VCI). In some embodiments, the subject has type 1, type 2, type 3, or type 4 diabetes. In some embodiments, the subject of the instructions has vascular dementia, with or without diabetes.

[0169] The kit includes a container having a composition that includes or consists essentially of a dose of a copper-lowering agent, such as triethylenetetramine disuccinate, preferably substantially pure triethylenetetramine disuccinate anhydride. The kit may further include a label or package insert on or associated with the container (or described as available online, in the cloud, or on a flash drive or another storage mechanism). The term "package insert" is used to refer to instructions that are customarily included in (or available online) commercial packages of therapeutic products, and include information regarding indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Suitable containers include, for example, bottles, blister packs, and the like. The container may be formed from a variety of suitable materials, including, for example, plastic. The container may be a package that includes a composition in the form of a tablet or capsule, the latter being one of the preferred forms when the copper antagonist or copper-lowering agent (e.g., triethylenetetramine disuccinate) is provided in, for example, a blister pack. The label or package insert indicates that the composition is used to treat a subject with diabetes and / or vascular dementia, and with (or suspected of having) cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration associated with excess or undesirable levels of hippocampal copper. In one embodiment, the instructions state that the copper antagonist or copper-lowering agent (e.g., triethylenetetramine disuccinate) is administered to a patient with diabetes and signs of cognitive impairment, cognitive decline, and / or dementia, or signs of brain neurodegeneration, and who is also receiving another therapeutic agent for cardiovascular disease, hypertension, diabetes, kidney disease, inflammation, depression, and / or dementia, etc. The instructions may refer to one or more of the doses or administration regimens described herein.

[0170] In some embodiments, the compound in the kit is selected from the group consisting of triethylenetetramine dihydrochloride, triethylenetetramine tetrahydrochloride, and triethylenetetramine disuccinate. In some embodiments, the triethylenetetramine disuccinate in the kit is triethylenetetramine disuccinate anhydride, preferably substantially pure triethylenetetramine disuccinate anhydride.

[0171] In one embodiment, the invention includes an article of manufacture that includes a package insert instructing a user to administer a copper antagonist compound (including one or more of the compounds described herein that include copper(I) and / or copper(II) chelators) capable of lowering or reducing copper levels or amounts (e.g., lowering copper levels, lowering total copper, and / or normalizing copper levels or amounts and / or copper metabolism) in a subject to a patient with a condition or disorder characterized by diabetes and dementia (or risk of dementia). The compound addresses excess copper in the hippocampus or elsewhere in the brain of the subject. The condition or disorder characterized by dementia in a patient with diabetes can be Alzheimer's disease. The condition or disorder characterized by dementia in a patient with diabetes can be vascular dementia.

[0172] In a further embodiment, the comorbidities in dementia patients treatable with compounds capable of lowering copper and / or normalizing copper metabolism (e.g., one or more of the copper chelators or copper-lowering agents described herein) are characterized by excess copper or reduced copper, respectively (such as type 2 diabetes or Alzheimer's). Thus, in a further embodiment, the comorbidities in dementia patients treatable with compounds capable of lowering and / or normalizing copper levels or amounts and / or copper metabolism (e.g., one or more of the copper chelators described herein) are characterized by copper deficiency (such as Alzheimer's). In another embodiment, the disease, condition, or disorder is selected from the group consisting of diabetes, Alzheimer's, Parkinson's, and Huntington's disease.

[0173] In another embodiment of the invention, the article of manufacture includes a container, a label, and a package insert. Suitable containers include, for example, bottles, blister packs, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a dose(s) of a copper antagonist or other copper depleting or removing agent composition (e.g., triethylenetetramine disuccinate) effective for treating one or more of the symptoms of cognitive impairment, cognitive decline, and / or dementia, or brain neurodegeneration, and conditions described herein. A label on or associated with the container indicates that the copper antagonist or other copper depleting or removing agent composition (e.g., triethylenetetramine disuccinate) is used for the treatments described herein. In certain embodiments, the patient has type 2 diabetes. In some embodiments, the patient has another form of diabetes. In some embodiments, the patient has type 1 or type 3 diabetes. In certain embodiments, the patient has heart failure. In certain embodiments, the patient has diabetic cardiomyopathy. In certain embodiments, the patient has left ventricular hypertrophy. The package insert, if desired, may include some or all of the clinical trial results that are found on clinicaltrials.gov or that are subsequently published, for example.

[0174] The evaluation of treatment with copper antagonists or other copper reducing or removing agents (e.g., triethylenetetramine disuccinate) can be performed by reference to the copper levels available in mammals, including humans. References herein to "elevated" in relation to the presence of copper levels include humans having at least about 10 mcg of free copper per dL of serum when measured. Measurement of free copper, which is equivalent to total plasma copper-ceruloplasmin bound copper, can be performed by a variety of procedures. A preferred procedure is disclosed in the Merck & Co. data sheet (www.Merck.com) for SYPRINE (trientine hydrochloride) capsules, a compound used for the treatment of Wilson's disease, in which a 24-hour urinary copper analysis is performed to determine the free copper (cooper) in serum by calculating the difference between the quantitatively measured total copper and ceruloplasmin-copper. EXAMPLES

[0175] Working Example The aim of this study was to evaluate hippocampal copper in type 2 diabetes (T2D) and sporadic Alzheimer's disease (sAD). Results from these experiments unexpectedly showed that brain hippocampal copper was significantly elevated in T2D, approximating the literature values ​​in Wilson's disease, whereas, in contrast, hippocampal copper levels in the brain of sporadic Alzheimer's disease patients were significantly deficient, supporting the use of copper-normalizing therapeutic approaches for the treatment of patients with diabetes who show or are at risk for cognitive impairment or decline, or who show signs of brain neurodegeneration or vascular dementia.

[0176] method Ethics. All experiments were performed in accordance with the relevant guidelines and regulations described below. Studies of postmortem human brain tissue were approved by the University of Manchester Research Ethics Committee. Informed consent for tissue collection for the T2D / control study was provided by the National Disease Research Interchange (NDRI, Philadelphia, PA), which hosts the Research Human Tissue and Organ Resources Program funded by the National Institutes of Health (www.ndriresource.org).

[0177] Human brain acquisition and sampling. Regional tissues (frontal cortex, temporal cortex, hippocampus, and associated meninges) from T2D cases and matched controls were obtained from the NDRI and transported to the University of Auckland, where they were dissected by a neuroanatomist and stored at -80°C until processing. Wet weight aliquots of 50±5 mg were dissected using a ceramic scalpel to avoid metal contamination, dried to constant weight in a centrifugal concentrator (Savant Speedvac™; Thermo-Fisher, Waltham, MA), and dry weight was measured by weighing on an analytical balance (DV215CD; Ohaus, Northamptonshire, UK). Dry weight measurements as used herein are preferred for measuring tissue metal levels in clinical laboratories (e.g., for measuring liver Cu for the diagnosis of WD) and are also commonly adopted for brain metal measurements (Xu J, Church Si, Patassini S, et al. Evidence for widespread, severe brain copper deficiency in Alzheimer's dementia Metallomics 2017;9:1106-19), and postmortem metal levels determined in this manner have been found to be stable, robust, and reproducible (Scholefield M, Church Si, Xu J, et al. Evidence that levels of nine essential metals in postmortem human-Alzheimer's-brain and ex vivo rat-brain tissues are unaffected by differences in postmortem delay, age, disease staging, and brain bank location. Metallomics 2020;12:952-62).

[0178] Diagnosis and severity. T2D cases were diagnosed by clinical history, whereas matched controls had no antemortem evidence of diabetes. Neither cases nor controls had a medical history or postmortem evidence of dementia or other brain disease. The presence of cognitive impairment was not recorded in the NDRI metadata, nor was it ruled out by formal mental status examination. For the AD / control study, the diagnosis and severity of sAD was determined by a consultant neuropathologist as described in Xu J,et al.Evidence for widespread,severe brain copper deficiency in Alzheimer's dementia Metallomics 2017;9:1106-19. Group characteristics for both the T2D / control and AD / control cohorts are shown in Table 1, and individual NDRI patient characteristics, including age and postmortem delay, are shown in Table 2. [Table 1] [Table 2]

[0179] Tissue digestion. Prior to digestion, all samples were briefly centrifuged at 2400 × g (Heraeus Pico 17 Centrifuge; Thermo Fisher Scientific, MA, US) to ensure that the tissue aliquot settled to the bottom of the tube. Concentrated nitric acid (A509 trace metal grade; Fisher, Loughborough, UK) and 5% Agilent internal standard mix (5183-4681; Agilent Technologies, Cheadle, UK) were combined to create the tissue digestion mix (Scholefield M, Church Si, Xu J, et al. Evidence that levels of nine essential metals in postmortem human-Alzheimer's-brain and ex vivo rat-brain tissues are unaffected by differences in postmortem delay, age, disease staging, and brain bank location. Metallomics 2020;12:952-62). Calibration standards were prepared using environmental calibration standard mix (Agilent Appropriate dilutions (Table SI) were prepared using 5% (5189-4688) and 2% (v / v) nitric acid digestion solution. For these dry weight analyses, 200 I.L1 of digestion solution was added to each sample, including two empty 2 ml microcentrifuge tubes as digestion blanks. The tube lids were punctured with a septum remover to prevent pressure build-up and then transferred to a room temperature Dri-Block DB3 heater (Techne, Staffordshire, UK). The temperature was set at 60 °C for 30 min and then further increased to 100 °C for 3.5 h. 100 1.1.1 of each sample or blank was then added to 5 ml of LC / MS grade water in a 15 ml Falcon tube (Greiner) and the samples were kept at room temperature until ICP-MS analysis.

[0180] ICP-MS. Metal concentrations were measured using an Agilent 7700x ICP-MS spectrometer equipped with a MicroMist nebulizer (Glass Expansion, Melbourne, Australia), a Scott double post-spray chamber, and nickel sample and skimmer cones. Samples were introduced into the spray chamber using an Agilent integrated autosampler (I-AS). Before each analysis, the sample tubing of the peristaltic pump was replaced to limit aberrant sample delivery to the nebulizer. To ensure consistency of system performance, ICP-MS system optimization and performance reports were generated in Agilent MassHunter Workstation software (G7201A, A.01.01) before each analysis.

[0181] Two collision cell gas modes were used to eliminate spectral interferences. All elements were analyzed in helium mode (5.0 ml / min He) except Se, which was analyzed in high energy helium mode (HEHe; 10 ml min'He) following Agilent's recommendations to reduce interference from polyatomic ion formation. Germanium and indium internal standards were analyzed in both modes. Integration times for relevant trace metals were 3 s for Se; 0.01 s for Fe; 0.03 s for Mn, Cu, and Zn; and 0.1 s for Na, Mg, K, and Ca. A multi-element method using serial dilutions of environmental calibration standards (Table 6; Agilent 5183-4688) was performed for each analytical batch. Internal standard calibration standard solutions of 50 μg / l and 5 μg / l were used as routine quality controls 1 and 2, respectively. The limits of quantification, limits of detection, and background equivalent concentrations for each trace metal analyzed in this report were generated automatically by Agilent MassHunter software. [Table 6-1]

[0182] Data Analysis. ICP-MS data sets were first exported to separate Microsoft Excel (2010) worksheets where they were corrected for sample weight and dilution, and then converted to units of mmol / kg or μmol / kg as appropriate. Means (±95% CI) were calculated and the significance of differences between groups was determined by unpaired Welch's (-tests) to account for unequal variances and sample sizes. Statistical calculations were performed using Prism v8.1.1 (GraphPad; La Jolla, CA). P values ​​less than 0.05 were considered significant. To identify cluster separations between T2D and AD metal datasets (Xu J, Church Si, Patassini S, et al. Evidence for widespread, severe brain copper deficiency in Alzheimer's dementia Metallomics 2017;9:1106-19), multivariate principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were applied using the R-platform MetaboAnalyst (www.metaboanalyst.ca). Chong J, Wishart DS, Xia J. Using MetaboAnalyst 4.0 for Comprehensive and Integrative Metabolomics Data Analysis. Curr Protoc Bioinformatics 2019;68(1):e86.

[0183] The mean of both T2D technical replicates was calculated for each regional analysis and was then used for all subsequent PCA and PLS-DA analyses. To assess the suitability of each data set for PCA, we first performed the Kaiser-Meyer-Olkin measure of sampling precision and Bartlett's goodness-of-fit test using SPSS version 23 (IBM; Armonk, NY). Prior to multivariate analysis, all metal data sets were mean-centered and divided by the standard deviation of each variable. Metals with a variable importance (VIP) score >I in the projection based on the PLS-DA model were considered to contribute to group separation.

[0184] result Results showed that hippocampal copper was substantially increased in six replicate T2D cases compared with six matched controls (Figure 1). This interpretation is further supported by similar results for copper in two consecutive technical replicate analyses (P=0.005 and P=0.007, respectively). The results provide strong evidence for the reproducibility of hippocampal Cu values ​​in this dataset (Figure 1; Tables 3 and 4). See National Academies of Sciences, Engineering, and Medicine. Reproducibility and Replicability in Science: Washington, DC: The National Academies Press; 2019. [Table 3-1] [Table 3-2] [Table 4]

[0185] The mean copper content of the hippocampus in cases was on average 2.2-fold higher compared with controls (394.8 vs. 180.2 μmol per kg dry tissue, respectively; Table 6). In contrast, none of the other eight elements were consistently different between cases and controls in technical replicate analyses (Fig. 1; Tables 3 and 4). In the first technical replicate, only Zn showed significant differences between cases and controls in any other region (frontal cortex, temporal cortex, and meninges) (Table 3). Cases and controls were matched for PMD (postmortem delay) and sex (Tables 3 and 4), whereas diabetic cases (70 years, 66-75 years [mean; range]) were slightly younger than controls (76 years, 69-78 years, p=0.011).

[0186] In the second technical replicate analysis, only the mean hippocampal copper (P=0.007) and Fe (P=0.044) levels were significantly increased in diabetic cases compared to controls (see Figure 2). As in the first analysis, the hippocampus showed the largest concentration difference between copper values ​​in cases and controls. However, for Fe, it was the meninges that showed the largest change in metal levels. The other physiological metals analyzed in this study did not show any statistically significant differences between the groups. [Table 6-2] Data is Cummings (WD) 19 and mean (range) and fold change of dry weight Cu concentrations from this study (T2D). Cumings 19It should be noted that Cu values ​​in were obtained using the sodium diethyldithiocarbamate method (not the reference method). Due to the different applications of the methodologies employed in these two studies, we determined that the fold change method is the preferred method to compare Cu values ​​between T2D and WD. The method used for T2D measurements is the current reference method. As only mean Cu concentrations for controls, and not individual brain concentrations, were provided, no measure of significance was obtained for Cumings' data. Only in the T2D hippocampal region were Cu values ​​significantly different between cases and controls (P = 0.005 and 0.007 in two consecutive technical repeat studies (Figures 1 and 2)).

[0187] Hippocampal copper showed P values ​​approaching the 3-sigma threshold for both technical replicates, consistent with robust data. See Colquhoun D. An investigation of the false discovery rate and the misinterpretation of p-values. R Soc Open Sci 2014;1(3):140216. Furthermore, subsequent statistical power analysis revealed that copper was the only metal to achieve a power of >0.9 in these studies. Thus, from a statistical validity standpoint, this body of evidence indicates that the finding that hippocampal copper is increased in T2D has a high level of importance and significance (see Table 7). [Table 7]

[0188] Because hippocampal copper was the predominantly variable essential metal in the T2D study, multivariate principal component analysis (PCA) was used to further characterize patterns within hippocampal tissue from AD (n = 9), T2D (n = 6), and control (n = 14) brains. Prior to performing PCA, the suitability of the analytical method was assessed. The Kaiser-Meyer-Olkin measure of sampling precision for the hippocampal dataset was 0.71, providing robust evidence for the validity of these datasets for PCA. Bartlett's test of sphericity was statistically significant (X 2 (36)=178.313, p<0.001), providing strong evidence for significant correlation between variables to support data reduction. Visual inspection of the scree plot revealed that the first two components explained 67.8% of the total variance, thus confirming the utility of two-dimensional PCA (Figure 4). As PCA plots comparing controls from both AD and T2D cohorts overlapped considerably, both control cohorts were combined and subsequently used as a single control group for comparison against AD and T2D (a pre-specified process). PCA plots for the hippocampus revealed a near-complete separation between AD and T2D, whereas there was considerable overlap between the T2D and control groups (Figure 3A), providing further robust evidence for differences in hippocampal metallomic profiles between AD and T2D.

[0189] To improve the discriminability of the data, partial least squares discriminant analysis (PLS-DA) was additionally performed on the same hippocampal dataset. The PLS-DA model showed a similar pattern to the PCA (Figure 3B), further supporting the main findings. Based on the PLS-DA model, a VIP score was generated to indicate the relative importance of each metal to group separation. For principal components 1 and 2; Na, Mn, Cu, and Fe all had VIP scores above 1, thus fulfilling the criteria to accurately identify these metals as reliable discriminators in our hippocampal dataset. Na achieved the highest VIP score in both components, whereas Cu achieved the third and second highest VIP scores in components 1 and 2, respectively (Figure 3C and Figure 3D; Table 5). [Table 5]

[0190] To allow for multi-region comparisons between PCAs, datasets derived from the frontal and temporal cortices and meninges were also analyzed by applying the same statistical approach as the hippocampal dataset. Importantly, no cluster separation was detected in any of the remaining three regions (Figures 5 and 6). These studies confirm that the distribution of PCA-derived hippocampal signals was substantially different from the distribution of signals arising from each of the other three regions (frontal cortex, temporal cortex, and meninges). The localization of signals in the hippocampus is also consistent with the localization of hippocampal damage in T2D. * * *

[0191] The invention described and claimed herein has many attributes and embodiments, including but not limited to those described, illustrated or referenced in this detailed description, which are not intended to be exhaustive, and the invention described and claimed herein is not limited to the features and embodiments identified in this detailed description, which is included for purposes of illustration only and not limitation.

[0192] All patents, publications, scientific articles, websites, and other documents and materials referenced or mentioned in this specification are indicative of the level of skill of one of ordinary skill in the art to which this invention pertains, and each such referenced document and material is incorporated herein by reference to the same extent as if it were individually incorporated by reference in its entirety or set forth in its entirety herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other referenced materials or documents. The reference to any application, patent, and publication in this specification is not, and should not be construed as, any form of admission or suggestion that they constitute valid prior art or form part of the general common general knowledge in any country in the world.

[0193] The specific methods and compositions described herein are representative of preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the scope of the invention as defined by the claims. Thus, for example, in each example and embodiment or example of the invention herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms herein. The methods and processes described herein as examples may suitably be performed with different sequences of steps, and are not necessarily limited to the sequence of steps set forth herein or in the claims. As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plurals unless the context clearly indicates otherwise. Under no circumstances shall this patent be construed as limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances shall this patent be construed as limited by any statements made by any examiner or any other official or employee of the Patent and Trademark Office. However, this does not apply where such a statement has been clearly and unconditionally agreed to and expressly adopted in the applicant's responsive written response.

[0194] The terms and expressions that have been used are used as terms of description rather than of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications may be made within the scope of the invention as defined in the claims. Thus, although the present invention has been specifically disclosed by preferred embodiments and features as required, it is understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are deemed to be within the scope of the invention as defined by the appended claims.

[0195] Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the invention are described in Markush form, those skilled in the art will recognize that the invention is also described in terms of any individual members or subgroups of members of the Markush group.

Claims

1. A pharmaceutical composition for treating cognitive impairment in a subject with type 2 diabetes, wherein the pharmaceutical composition comprises a compound capable of reducing copper in the subject, and wherein urinary copper increases after administration of the composition, and cognitive impairment in the subject is reduced.

2. The pharmaceutical composition according to claim 1, wherein the compound can reduce elevated copper levels in the hippocampus of the subject.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises triethylenetetramine and a pharmaceutically acceptable carrier, flow promoter, diluent, or excipient.

4. The pharmaceutical composition according to claim 3, wherein the triethylenetetramine is in the form of a pharmaceutically acceptable salt.

5. The pharmaceutical composition according to claim 4, wherein the triethylenetetramine salt is selected from the group consisting of triethylenetetramine dihydrochloride, triethylenetetramine tetrahydrochloride, and triethylenetetramine disuccinate.

6. The pharmaceutical composition according to claim 1, wherein the compound is triethylenetetraminedisucinate.

7. The pharmaceutical composition according to claim 1, wherein the triethylenetetraminedisuccinate is a crystalline form of triethylenetetraminedisuccinate.

8. The pharmaceutical composition according to claim 1, wherein the triethylenetetraminedisuccinate is triethylenetetraminedisuccinate anhydride.

9. The pharmaceutical composition according to claim 1, wherein the subject exhibits signs of cerebral degeneration, and one or more signs of cerebral degeneration are improved.

10. The pharmaceutical composition according to claim 1, wherein the subject has or is at risk of having cognitive decline and / or dementia, and the cognitive decline and / or dementia in the subject is improved.

11. The pharmaceutical composition according to claim 1, wherein the subject has impaired spatial memory or the ability to remember directions, locations, and orientations, and the subject's spatial memory and / or ability to remember directions, locations, and / or orientations is improved.

12. The pharmaceutical composition according to claim 1, characterized in that it is administered in combination with one or more further therapeutic agents selected from anti-inflammatory agents, agents for treating cardiovascular disease, agents for treating hypertension, agents for treating kidney disease, and agents for treating type 2 diabetes.

13. The pharmaceutical composition according to claim 12, wherein the agent for treating type 2 diabetes is selected from the group consisting of alpha-glucosidase inhibitors, biguanides, dopamine agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 receptor agonists, meglitinides, sodium-glucose transporter (SGLT) 2 inhibitors, sulfonylureas, and thiazolidinediones.

14. The pharmaceutical composition according to claim 1, wherein the subject of the test is a human.

15. The pharmaceutical composition according to claim 1, characterized in that it is administered orally in the form of a capsule or tablet.

16. The pharmaceutical composition according to claim 1, characterized in that the compound is triethylenetetramine dihydrochloride and is administered in an amount of approximately 1200 mg daily.

17. The pharmaceutical composition according to claim 1, characterized in that the compound is triethylenetetraminedisuccinate, and a daily dose of triethylenetetraminedisuccinate ranging from about 2400 mg / day to about 3000 mg / day is administered.

18. The pharmaceutical composition according to claim 1, characterized in that the compound is triethylenetetraminedisuccinate and is administered in an amount of approximately 2800 mg daily.

19. The pharmaceutical composition according to claim 16, characterized in that the 1200 mg is administered in divided doses of 600 mg as BID, in divided doses of 400 mg as TID, or in divided doses of 300 mg as QID.

20. The pharmaceutical composition according to claim 18, characterized in that the aforementioned 2800 mg is administered in divided doses.

21. A kit for therapeutic treatment of cognitive impairment in subjects with type 2 diabetes, comprising: a) a pharmaceutical composition comprising a compound capable of lowering copper levels and / or normalizing copper metabolism in subjects; and b) instructions for use in the therapeutic treatment or prevention of cognitive impairment in subjects with type 2 diabetes.

22. The kit according to claim 21, wherein the compound is selected from the group consisting of triethylenetetramine dihydrochloride, triethylenetetramine tetrahydrochloride, and triethylenetetramine disuccinate.

23. The kit according to claim 22, wherein the triethylenetetramindisuccinate is triethylenetetramindisuccinate anhydrous.

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