WO2017213524A1

WO2017213524A1 - Treatment of neurodegenerative disorders

Info

Publication number: WO2017213524A1
Application number: PCT/NZ2017/050075
Authority: WO
Inventors: Garth Cooper
Original assignee: Garth Cooper
Priority date: 2016-06-10
Filing date: 2017-06-09
Publication date: 2017-12-14

Abstract

Pharmaceutical compositions and methods for the treatment of subjects who have, or are at risk for, Alzheimer's disease. The composition comprises triethylenetetramine succinate compounds, alone or in combination with agents to reduce the amount or concentration of one or more encephalic glucose, sorbitol or fructose, such as amylin, amylin analogue, a GLP-1 agonist or a selective dipeptidyl peptidase IV inhibitor.

Description

TREATMENT OF NEURODEGENERATIVE DISORDERS

FIELD OF THE INVENTION

[0001] The present invention relates generally to compounds, compositions and methods of treatment, and include compounds, compositions and methods for treating Alzheimer's disease, for improving physiological and/or neurological deficits associated with Alzheimer's disease.

BACKGROUND TO THE INVENTION

[0002] The following includes information that may be useful in understanding the present invention . It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or clai med invention, or that any publication or document that is specifically or implicitly referenced is prior art.

[0003] Alzheimer's disease (AD) is an age-related neurodegenerative disease associated with pathological characteristics of senile plaques and neurofibrillary tangles. It is the most common form of dementia in humans, with a progression from episodic memory problems to a general decline in cognitive function . In 2013, approximately 44 million people were estimated to be affected by dementia.

[0004] A number of mechanisms have been proposed as causative of AD. As yet, however, no curative treatment with proven disease modifying effects exists.

[0005] Current treatments for AD are directed to treating cognitive and behavioural symptoms. Two types of drugs are currently used to treat cognitive symptoms of AD;

cholinesterase inhibitors, which provide the neurotransmitter acetylcholine that is commonly depleted in the AD brain and thereby purportedly i mprove cell-to-cell communication; and memantine hydrochloride (Namenda™), which is believed to inhibit signal transduction of N- methyl-d-aspartate (N MDA) receptors, and reportedly slows the progression of symptoms in moderate to severe AD.

[0006] Other medications, such as antidepressants, are sometimes used to help control the behavioural symptoms associated with AD.

[0007] There are no known approved treatments that are directed to the cause of AD, the underlying dysfunction or the resulting cell and tissue damage. Clearly there is a need for compounds and methods that limit or prevent damage associated with or causative of AD, as well as damage to organelles, cells and tissues that occurs as a consequence of AD.

[0008] There is also a need for compounds and methods that limit or prevent damage to cells and tissues that occurs directly or indirectly as a result of necrosis and/or inappropriate apoptosis associated with or causative of AD. Agents and methods that maintain neuronal and glial cell integrity represent novel protective agents with utility in limiting AD and AD-related dysfunction .

[0009] Copper chelation, optionally in combination with treatment to lower encephalic glucose, encephalic sorbitol, and/or encephalic fructose, with the copper chelating agent triethylenetetramine succinate is proposed herein to treat AD.

[0010] U.S. Patent No. 7582796, issued on 1 September 2009, provides methods of synthesising triethylenetetramine copper chelators, including representative

triethylenetetramine succinates suitable for use in the present invention .

[0011] It is an object of the invention to go at least some way to fulfilling these needs and provide other related advantages, and/or to at least provide the public with a useful choice. Those skilled in the art will recognize further advantages and benefits of the invention after reading the disclosure.

SUMMARY OF THE INVENTION

[0012] The invention described and claimed herein have many attributes and embodiments including, but not li mited to, those set forth or described or referenced in this Summary. It is not intended to be all-inclusive and the invention described and clai med herein are not limited to or by the features or embodiments identified in this Summary, which is included for purposes of illustration only and not restriction .

[0013] The present invention relates generally to compounds, compositions and methods for treating Alzheimer's disease (AD).

[0014] The present invention is directed in part to the treatment of AD by

administration to a mammalian subject in need thereof an effective amount of a

triethylenetetramine succinate.

[0015] In various embodiments, the triethylenetetramine succinate binds Cu(II), for example, is a triethylenetetramine succinate that is specific for Cu(II) over Cu(I) .

Particularly contemplated triethylenetetramine succinates include triethylene 2,2,2 tetramine succinates, triethylene 2,3,2 tetramine succinates and triethylene 3,3,3 tetramine succinates, as well as active metabolites, derivatives, and prodrugs thereof.

[0016] Useful triethylenetetramine succinates include triethylenetetramine disuccinate, such as triethylenetetramine disuccinate anhydrate. [0017] In certain embodiments, the triethylenetetramine succinate is crystalline triethylenetetramine succinate, for example a crystalline triethylenetetramine disuccinate such as a crystalline triethylenetetramine disuccinate anhydrate.

[0018] In certain embodiments, the triethylenetetramine succinate is a polymorph of triethylenetetramine succinate, such as a triethylenetetramine disuccinate polymorph having a differential scanning calori metry (DSC) extrapolated onset/peak melting temperature of from between about 170°C to about 190°C.

[0019] In certain embodiments, the triethylenetetramine succinate is a crystalline triethylenetetramine succinate, for example a triethylenetetramine disuccinate such as a triethylenetetramine disuccinate anhydrate, in the form of a crystal having alternating layers of triethylenetetramine molecules and succinate molecules.

[0020] In one particularly contemplated embodiment, the method is a method of treating AD by administering to a mammalian subject in need thereof one or more of a dosage form comprising from about 250 mg to about 280mg of triethylenetetramine disuccinate anhydrate. For example, the method comprises administering to a mammalian subject in need thereof one or more of a dosage form comprising about 280mg of triethylenetetramine disuccinate anhydrate.

[0021] Optionally, the method additionally comprises administration to the subject of an effective amount of an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject. [0022] In various embodiments, the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is selected from the group comprising amylin, an amylin analogue, a GLP-1 agonist, and a selective dipeptidyl peptidase (DPP-IV) inhibitor.

[0023] In one example, the amylin analogue is Symlin . [0024] In various examples, the GLP-1 agonist is exenatide, liraglutide, lixisenatide, albiglutide, or dulaglutide, or any combination of two or more thereof. [0025] In various examples, the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin .

[0026] In one example, the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising alogliptin, linagliptin, saxagliptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia .

[0027] When used in combination, administration of the triethylenetetramine succinate and of the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject may be simultaneous, sequential, or separate.

[0028] In still further embodi ments, methods are provided for treating AD by administering an effective amount of a triethylenetetramine succinate in the form of a pharmaceutical composition .

[0029] In one embodi ment, the pharmaceutical composition additionally comprises one or more agents effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in a mammalian subject.

[0030] In various embodiments, the pharmaceutical composition comprises one or more triethylenetetramine succinates additionally comprising or formulated to be

administered in conjunction in with an agent effective to reduce the amount or

concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject, wherein the one or more pharmaceutical compositions comprises a pharmaceutically acceptable carrier or diluent.

[0031] In various embodiments, the pharmaceutical composition comprises triethylenetetramine disuccinate, such as triethylenetetramine disuccinate anhydrate. [0032] In certain embodiments, the pharmaceutical composition comprises crystalline triethylenetetramine succinate, for example a crystalline triethylenetetramine disuccinate such as a crystalline triethylenetetramine disuccinate anhydrate.

[0033] In certain embodiments, the pharmaceutical composition comprises a polymorph of triethylenetetramine succinate, such as a triethylenetetramine disuccinate polymorph having a differential scanning calorimetry (DSC) extrapolated onset/peak melting temperature of from between about 170°C to about 190°C. [0034] In certain embodiments, the pharmaceutical composition comprises a crystalline triethylenetetramine succinate, for example a triethylenetetramine disuccinate such as a triethylenetetramine disuccinate anhydrate, in the form of a crystal having alternating layers of triethylenetetramine molecules and succinate molecules. [0035] In certain embodiments, the pharmaceutical composition comprises a crystalline triethylenetetramine succinate in the form of a crystal having alternating layers of triethylenetetramine molecules and succinate molecules are also provided, including pharmaceutical compositions having crystalline triethylenetetramine disuccinate anhydrate in the form of a crystal having alternating layers of triethylenetetramine molecules and succinate molecules.

[0036] In certain embodiments, the crystalline triethylenetetramine disuccinate has a C 2/c (no. 15) space group with measured unit cell di mensions of a= 14.059(5) A, b = 9.169(5) A, c= 13.647(5) A, and β = 92.47(0) A, is provided. Cell volume is 1757.56(130) [0037] In certain embodiments, the crystalline triethylenetetramine disuccinate has the structure defined by the co-ordinates of Tables 1A-1C.

[0038] In the context of the invention, AD includes tissue damage or degeneration associated with, caused by, or causative of AD, including damage or degeneration i n which free radical mediated oxidative injury is involved, and damage or degeneration associated with AD in which cells inappropriately undergo apoptosis. Thus, treatment or amelioration of symptoms of AD, including cognitive and behavioural symptoms, are particularly

contemplated.

[0039] In various embodiments, the mammalian subject is selected from the group consisting of a human, a domestic and farm animal, and zoo, sports, or pet animal, such as a dog, a horse, a cat, a sheep, a pig, a cow, or a deer. In a particularly contemplated example, the mammalian subject is a human .

[0040] In various embodiments, the mammalian subject is a non-diabetic subject, for example, a non-diabetic human subject, or a human subject who is not undergoing treatment for diabetes. In various embodiments, the mammalian subject does not suffer from Wilson's disease.

[0041] In one embodi ment, the reduction in the amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose is by a statistically significant amount. In one example, the reduction in the amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose is to an amount or concentration substantially equivalent to the amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose, respectively, found in a mammalian subject of the same species not suffering from AD. [0042] In one embodi ment, the reduction in the amount or concentration of encephalic glucose is to an amount or concentration substantially equivalent to the amount or concentration of encephalic glucose found in a mammalian subject of the same species not suffering from AD.

[0043] Diagnostic or prognostic methods for determining the presence of or likelihood of developing AD are also provided.

[0044] In one aspect, the invention relates to a method of treating AD in a mammalian subject in need thereof or suspected of being in need thereof, the method comprising :

analysing an encephalic sample from said subject to determine the amount or concentration of glucose, fructose, or sorbitol in the sample, and

comparing the amount or concentration of glucose, fructose, or sorbitol in the sample to the amount or concentration of glucose, fructose, or sorbitol found in a mammalian subject of the same species not suffering from AD,

wherein an elevated amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose in the sample is indicative of an increased risk of developing AD or of the presence of AD,

and administering to a mammalian subject determi ned to be at an increased risk of developing AD or suffering from AD an effective amount of a triethylenetetramine succinate.

[0045] In one aspect, the invention relates to a method of assessing a mammalian subject's risk of developing AD which comprises:

wherein an elevated amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose in the sample is indicative of an increased risk of developing AD, and optionally administering to the mammalian subject an effective amount of a triethylenetetramine succinate. [0046] In one embodi ment, an elevated concentration of encephalic glucose in a sample from a human subject indicative of an increased risk of developing AD is above about 10 pmol glucose/g wet weight.

[0047] In a further aspect, the invention relates to the use of a triethylenetetramine succinate in the treatment of AD.

[0048] In a further aspect, the invention relates to the use of a triethylenetetramine succinate in the preparation of a medicament for the treatment of AD.

[0049] In various embodiments, the invention concerns pharmaceutical compositions containing such agents, articles and kits and delivery devices containing such agents, and tablets and capsules and formulations comprising such agents or compositions. Particularly contemplated are articles, kits and delivery devices enabling the separate, sequential or simultaneous administration of (a) one or more triethylenetetramine succinates, and of (b) the one or more agents effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in a mammalian subject. [0050] In certain embodi ments, the pharmaceutical composition comprises substantially pure triethylenetetramine succinate, such as substantially pure

triethylenetetramine disuccinate anhydrate.

[0051] Pharmaceutical compositions also comprise a pharmaceutically acceptable carrier or diluent. [0052] Metabolites include, for example, acetylated metabolites, such as N-acetyl triethylenetetramine (e.g ., monoacetyl-triethylenetetramine) . Derivatives include, for example, PEG-modified triethylenetetramines.

[0053] The one or more triethylenetetramine succinates may be administered as compositions in amounts, for example, that are effective to chelate encephalic Cu(II) . Such compositions include, for example, tablets, capsules, solutions and suspensions for parenteral and oral delivery forms and formulations.

[0054] In various embodiments, the therapeutic methods described herein are carried out in conjunction with dietary or lifestyle modifications. The simplest of these regi mens can be the provision to a subject with AD of motivation to i mplement such a lifestyle change, for example, dietary adjustments to reduce foods contributing to elevated brain urea concentration observed in AD, such as high protein foods. [0055] These and other aspects of the invention, which are not limited to or by the information in this Summary, are provided below.

[0056] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational nu mbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).

[0057] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth .

[0058] In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention . Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

[0059] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

BRIEF DESCRIPTION OF THE DRAWINGS [0060] The invention will now be described by way of example only and with reference to the drawings in which :

Figure 1 shows regional A) mean glucose levels, and B) brain copper levels, in the cerebellum (CB), cingulate gyrus (CG), entorhinal cortex (ENT), hippocampus (HP), motor cortex (MCx), middle temporal gyrus (MTG), and sensory cortex (SCx) of patients with AD (n = 7-9/region; on right) and control patients (n = 7-9/region on left) . DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention utilises a specific agent for copper chelation, a triethylenetetrannine succinate, to treat or prevent AD. The triethylenetetramine succinates useful herein have advantageous physicochemical properties rendering them particu larly suitable for use in the treatment of AD.

[0062] As presented in the Examples, the applicant has demonstrated that 1) the AD brain is copper-deficient, and 2) levels of glucose, sorbitol and fructose are elevated across many regions of the AD brain .

[0063] Without wishing to be bound by any theory, the applicant believes that elevated levels of glucose, fructose and sorbitol in the brain are likely to be toxic via the formation of advanced glycation endproducts (AGEs) such as N-epsilon-carboxy-methylysine (CML) .

[0064] The formation of AGEs in the brain creates pathogenic copper (II) binding sites in the extracellular matrix (ECM) (Kamalov et al ., 2015. Org. Biomol. Chem. 14 : 13(1) : 3058-3063) . Copper bound to AGEs such as CML is catalytically active and therefore toxic.

[0065] The applicant believes that ECM-bound copper (II) causes deficient neuronal copper uptake via suppression of membrane-bound copper uptake mediated by copper transporter CTRl and intracellular copper transport via copper chaperones (see Zhang et al ., 2014 Cardiovascular Diabetology 13 : 100) . [0066] It has been shown that CML-bound copper itself catalyses glucose-mediated AGE formation in a feed-forward reaction (Brings et al ., 2015. Biochim. Biophys. Acta 1852(8) : 1610-1618) .

[0067] Physiological copper cations are crucial to the function of enzymes involved in processes including tissue antioxidant defence, suppression of inflammation and effective utilisation of metabolic fuels. Enzymes that are targeted and repaired by copper chelation include superoxide dismutase I (SOD1), superoxide dismutase III (SOD3), cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit II (COII) .

[0068] Reduction of copper available to be incorporated into these enzymes as seen in the AD brain results in i mpaired function of the enzymes, which may result in

mitochondrial dysfunction . For example, a reduction in cytochrome c oxidase activity leads to increased electron leaking from the mitochondrial and oxidative stress. [0069] The use of copper chelation, optionally together with glucose-lowering treatment, inhibits binding of copper to AGEs such as CML, thus reducing AGE-bound copper-induced suppression of copper transport in the brain . The methods of the invention thus suppress AGE formation, ameliorate copper deficiency and restore enzyme and mitochondrial function in the brain .

[0070] As used herein, a "copper antagonist" is a pharmaceutically acceptable compound that binds or chelates copper in vivo for removal . Copper chelators are specifically contemplated copper antagonists. For example, copper (II) chelators, and copper (II) specific chelators {i.e., those that preferentially bind copper (II) over other forms of copper such as copper (I)), are particularly contemplated.

[0071] As used herein, "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids the like. When a compound is basic, for example, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. In the context of triethylenetetramine succinates, anhydrous disuccinates are specifically contemplated .

[0072] As used herein, "preventing" means preventing in whole or in part, or ameliorating or controlling.

[0073] As used herein, "substantially pure" means substantially free from any other compounds. In one embodi ment, it contains less than 10% i mpurities, for example, less than about 5% i mpurities, and in a specifically contemplated example, less than about 1% i mpurities. The product thus formed is also in one embodiment substantially pure, i .e., contains less than 10% i mpurity, for example, less than 5% impurity, and in a specifically contemplated example, less than 1% impurity. Particularly contemplated herein is a substantially pure anhydrous crystalline form of triethylenetetramine disuccinate. The term "substantially pure" when used in reference to a particular polymorphic form means that a sample of the relevant anhydrous crystalline form of triethylenetetramine disuccinate contains more than 90% of a single polymorphic form, for example, more than 95% of a single polymorphic form, and in a specifically contemplated example, more than 99% of a single polymorphic form. [0074] As used herein, a "therapeutically effective amount" in reference to the compounds or compositions of the instant invention refers to the amount sufficient to induce a desired biological, pharmaceutical, or therapeutic result. That result can be alleviation of the signs, symptoms, or causes of a disease or disorder or condition, or any other desired alteration of a biological system . In one aspect of the present inventions, the result will involve the prevention, decrease, or reversal of AD, in whole or in part, and prevention and/or treatment of related conditions, including those referenced herein .

[0075] As used herein, the term "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those prone to having the disorder, or those diagnosed with the disorder, or those in which the disorder is to be prevented .

[0076] As used herein, encephalic glucose refers the glucose present in the brain and/or encephalic cavity of a mammalian subject, and particularly contemplates glucose present in one or more tissues or regions of the brain . Methods to determi ne the amount or concentration of encephalic glucose are known in the art, and exemplary methods are provided herein in the Examples.

[0077] As used herein, encephalic fructose refers the glucose present in the brain and/or encephalic cavity of a mammalian subject, and particularly contemplates fructose present in one or more tissues or regions of the brain . Methods to determine the amount or concentration of encephalic fructose are known in the art, and exemplary methods are provided herein in the Examples.

[0078] As used herein, encephalic sorbitol refers the glucose present in the brain and/or encephalic cavity of a mammalian subject, and particularly contemplates sorbitol present in one or more tissues or regions of the brain . Methods to determine the amount or concentration of encephalic sorbitol are known in the art, and exemplary methods are provided herein in the Examples.

[0079] It will be appreciated that in specifically contemplated embodiments, agents that are effective to lower encephalic glucose, sorbitol, or fructose without a concomitant risk of hypoglycaemia are used . Representative examples of such agents are discussed below.

Amylin and amylin analogues

[0080] Amylin is a small peptide hormone released into the bloodstream by the pancreatic β-cells after a meal, and reportedly modulates blood glucose by slowing gastric emptying, promoting satiety, and inhibiting inappropriate secretion of glucagon . Amylin analogues, such as symlin (Pramlintide™, AstraZeneca), are used to augment endogenous amylin, or to replace amylin's function in diabetics who do not naturally produce amylin .

GLP-1 agonists [0081] GLP-1 is a naturally-occurring peptide that is released within minutes of eating a meal . It has been reported to suppress glucagon secretion from pancreatic alpha cells and sti mulate insulin secretion by pancreatic beta cells. GLP-1 receptor agonists are generally used in the treatment of type 2 diabetes. Representative GLP-1 agonists suitable for use in the methods and compositions described herein include exenatide

(Byetta™/Bydureon™, AstraZeneca, approved in 2005/2012), liraglutide (Victoza™,

Saxenda™, Novo Nordisk, FDA approved 2010), lixisenatide (Lyxumia™, Zealand Pharma & Sanofi, approved in EU 2013), albiglutide (Eperzan™, Tanzeum™, GSK, FDA approved in 2014), and dulaglutide (Trulicity™, Eli Lily, FDA approved in 2014) .

Selective dipeptidyl peptidase (DPP-IV) inhibitors [0082] Dipeptidyl peptidase (DPP-IV) inhibitors have been reported to reduce glucagon and blood glucose levels via increasing insulin levels. Representative DPP-IV inhibitors suitable for use in the method and compositions described herein include

Sitagliptin (FDA approved 2006, marketed by Merck & Co. as Januvia™), Vildagliptin (EU approved 2007, marketed in the EU by Novartis as Galvus™), Saxagliptin (FDA approved in 2009, marketed as Onglyza™), Linagliptin (FDA approved in 2011, marketed as Tradjenta™ by Eli Lilly Co and Boehringer Ingelhei m), Anagliptin (approved in Japan in 2012, marketed by Sanwa Kagaku Kenkyusho Co., Ltd. and Kowa Company, Ltd .), Teneligliptin (a pproved in Japan in 2012), Alogliptin (FDA approved 2013, marketed by Takeda Pharmaceutical Company), Trelagliptin (approved for use in Japan in 2015), Gemigliptin (LG Life Sciences), Dutogliptin (Phenomix Corporation), and Omarigliptin (approved in Japan in 2015, developed by Merck & Co.).

Copper binding compounds

[0083] The copper binding compound triethylenetetramine succinate has been determined by the applicant to be particularly suitable for treating AD, being effective in selectively chelating copper (II), and having advantageous physicochemical characteristics that make it particularly suitable for prescription for and administration to subjects having or at risk of developing AD. This is at least in part due to the advantageous stability of triethylenetetramine succinates, particularly triethylenetetramine disuccinate anhydrate. This advantageous stability obviates the need for either cool storage or storage in lightproof containers. It will be appreciated by those skilled in the art on readi ng this specification that such attributes are particularly useful in the context of administration, for example self- administration, to subjects suffering from or at risk of developing AD, where compliance to a treatment regimen is frequently a concern .

[0084] Suitable triethylenetetramine succinates include triethylene 2,2,2 tetramine succinates, triethylene 2,3,2 tetramine succinates, and triethylene 3,3,3 tetramine succinates, and particularly the disuccinate anhydrates thereof, as well as active

metabolites, derivatives, and prodrugs thereof.

[0085] Triethylene 2,2,2 tetramine (also known under IUPAC terminology as Ν,Ν'- bis(2-aminoethyl)ethane-l,2-diamine) has the following formula (I)

(I), while triethylene 2,3,2 tetramine (also known under IUPAC terminology as /V,/V'-bis(2- aminoethyl)propane-l,3-diamine) has the following formula (II)

(Π), and triethylene 3,3,3 tetramine (also known under IUPAC terminology as /V,/V'-bis(3- aminopropyl)propane-l,3-diamine) has the following formula (III)

(III) .

[0086] Crystalline forms of triethylenetetramine succinates, such as

triethylenetetramine disuccinate anhydrate, are particularly contemplated . A representative crystalline triethylenetetramine disuccinate has the characteristics set out in Table 1A - 1C below. Table 1A

Crystallographic and refinement data for triethylenetetramine disuccinate formula sum Ci4 H34 N4 Os

formula weight 386.44

measurement temperature 84(2) K

measurement device Bruker SMART CCD

wavelength 0,71076 A (Mo-Ka-radiation)

crystal system monoclinic

space group C 2/c (no. 15)

unit cell dimensions a = 14.059(5) A

b = 9.169(5) A

c = 13.647(5) A

β = 92.47(0) °

cell volume 1757.56(130) A³

Z 4

density, calculated 1.007 g/cm³

absorption coefficient μ 0.077 mm ¹

F(000) 584

Θ range 2,65 - 25,66°

h min, h max^ kmin, kmax^ Imin, I max -17, 16; -6, 11; -14, 16

reflections measured 4899 [R(int) = 0,0322]

independent reflections 1653

observed reflections [I>2s(i)] 1455

data / restraints / parameters 1653 / 0 / 118

Goodness-of-fit at F² 1.053

R indices [I > 2 sigma(I)] Rl = 0.0366, wR2 = 0.0948

R indices (all data) Rl = 0.0431, wR2 = 0.0987

largest diff. peak / hole 0,266 / -0,260 e'A ³

Table IB

Triethylenetetramine disuccinate atomic coordinates and

isotropic displacement parameters (in A²)

H1B 0.27720 0.19900 0.37230 0.01500

C2 0.2803(1) 0.18094(16) 0.22253(10) 0.0122(3)

H2A 0.33450 0.11790 0.21290 0.01500

H2B 0.22430 0.11980 0.22590 0.01500

C3 0.2663(1) 0.19924(16) 0.04178(9) 0.0124(3)

H3A 0.22310 0.11710 0.04510 0.01500

H3B 0.32940 0.16200 0.03010 0.01500

C4 0.0209(1) 0.46728(16) 0.12805(9) 0.0113(3)

C5 -0.0467(1)0.33720(16) 0.11701(10) 0.0129(3)

H5A -0.08810 0.35300 0.05930 0.01500

H5B -0.08650 0.33540 0.17330 0.01500

C6 -0.00004(10) 0.18713(16) 0.10792(10) 0.0134(3)

H6A 0.04010 0.18650 0.05180 0.01600

H6B 0.03980 0.16740 0.16620 0.01600

C7 -0.07602(10) 0.06870(16) 0.09545(10) 0.0114(3)

Nl 0.39566(8) 0.31058(14) 0.33899(8) 0.0117(3)

H1C 0.40020 0.35710 0.39620 0.01800

HID 0.41260 0.37040 0.29140 0.01800

HIE 0.43390 0.23320 0.34110 0.01800

N2 0.26795(8) 0.28179(14) 0.13633(8) 0.0109(3)

H2C 0.21310 0.33160 0.14060 0.01300

H2D 0.31610 0.34660 0.13740 0.01300

01 -0.01814(7) 0.59176(11) 0.12409(7) 0.0154(3)

02 0.10919(7) 0.44737(11) 0.14249(7) 0.0153(3)

03 -0.12598(8) 0.06534(12) 0.01693(7) 0.0188(3)

04 -0.08767(7) -0.01952(12) 0.16553(7) 0.0178(3)

TABLE lC

Triethylenetetramine disuccinate anisotropic displacement parameters (in A²)

CI 0.0120(7) 0.0130(8) 0.0129(7) 0.0010(6) 0.0011(5) 0.0010(6)

C2 0.0135(7) 0.0099(7) 0.0130(7) -0.0003(5) -0.0007(5) 0.0017(5)

C3 0.0133(7) 0.0113(7) 0.0125(7) 0.0000(5) -0.0007(5) -0.0024(6)

C4 0.0132(7) 0.0121(8) 0.0087(6) 0.0002(6) 0.0016(5) -0.0012(5)

C5 0.0106(7) 0.0123(8) 0.0158(7) -0.0010(6) 0.0007(5) -0.0002(5) C6 0.0125(7) 0.0122(8) 0.0154(7) -0.0013(6) 0.0011(5) 0.0006(5)

C7 0.0128(7) 0.0092(7) 0.0123(6) 0.0019(5) 0.0018(5) -0.0011(5)

N l 0.0136(6) 0.0111(6) 0.0104(5) 0.0010(5) -0.0008(4) -0.0006(4)

N2 0.0108(6) 0.0103(6) 0.0115(6) -0.0003(5) -0.0005(4) 0.0000(5)

01 0.0139(5) 0.0106(6) 0.0216(5) 0.0007(4) -0.0008(4) -0.0006(4)

02 0.0107(5) 0.0131(6) 0.0219(5) 0.0001(4) 0.0001(4) -0.0026(4)

03 0.0241(6) 0.0185(6) 0.0132(5) -0.0079(4) -0.0044(4) 0.0033(4)

04 0.0206(6) 0.0174(6) 0.0150(5) -0.0066(4) -0.0032(4) 0.0055(4)

[0087] The triethylenetetramine succinates may be made using any of a variety of chemical synthesis, isolation, and purification methods known in the art. Synthetic methods of particular use are those described in U .S. Patent No. 7582796, issued on 1 September 2009, incorporated herein by reference in its entirety. [0088] In still further embodi ments, methods are provided for treating AD by administering one or more triethylenetetramine succinates in the form of a pharmaceutical composition . Thus, pharmaceutical compositions are also provided comprising one or more triethylenetetramine succinates, in combination with a pharmaceutically acceptable carrier or diluent. [0089] Triethylenetetramine succinates useful in the methods described herein also include triethylenetetramine succinates that have been pre-complexed with a non-copper metal ion prior to administration for therapy. Metal ions used for pre-complexing have a lower association constant for the triethylenetetramine succinate than that of copper. For example, a metal ion for pre-complexing a triethylenetetramine succinate that chelates Cu²⁺ is one that has a lower binding affinity for the triethylenetetramine succinate than Cu²⁺. Specifically contemplated metal ions for precomplexing include calcium (e.g., Ca²⁺), magnesium (e.g., Mg²⁺), chromium (e.g., Cr²⁺ and Cr³⁺), manganese (e.g., Mn²⁺), zinc (e.g., Zn²⁺), selenium (e.g ., Se⁴⁺), and iron (e.g., Fe²⁺ and Fe³⁺) . In certain examples, metal ions for precomplexing are calcium, zinc, and iron . Other metals include, for example, cobalt (e.g., Co²⁺), nickel (e.g., Ni²⁺), silver (e.g., Ag¹⁺), and bismuth (e.g., Bi³⁺) . Metals are chosen with regard, for example, to their relative binding to the

triethylenetetramine succinate, and relative to toxicity and the dose of the

triethylenetetramine succinate to be administered .

[0090] Also encompassed are metal complexes comprising triethylenetetramine succinates and non-copper metals (that have lower binding affinities than copper for the triethylenetetramine succinate) and one or more additional ligands than typically found in complexes of that metal . These additional ligands may serve to block sites of entry into the complex for water, oxygen, hydroxide, or other species that may undesirably complex with the metal ion and can cause degradation of the triethylenetetramine succinate. For example, copper complexes of triethylenetetramine have been found to form

pentacoordinate complexes with a tetracoordinated triethylenetetramine and a chloride ligand when crystallized from a salt solution rather than a tetracoordinate Cu²⁺

triethylenetetramine complex. In this regard, 219 mg of triethylenetetramine. 2 HCI were dissolved in 50 ml, and 170 mg of CuC ^■ 2H2O were dissolved in 25 ml ethanol (95%) . After addition of the CuC solution to the triethylenetetramine solution, the colour changed from light to dark blue and white crystals precipitated . The crystals were dissolved by addition of a solution of 80 mg NaOH in 15 ml H2O. After the solvent was evaporated, the residue was dissolved in ethanol, and two equivalents of ammonium-hexafluorophosphate were added . Blue crystals could be obtained after reduction of the solvent. Crystals were found that were suitable for x-ray structure determination . X-ray crystallography revealed a [Cu(triethylenetetramine)CI] complex. Other coordinated complexes may be formed from or between triethylenetetramine succinates, for example, copper chelators (such as Cu2+ chelators, spermidine, spermine, tetracyclam, etc.), particularly those subject to

degradative pathways such as those noted above, by providing additional complexing agents (such as anions in solution, for example, I^", Br, F^~, (SO4)^2", (CO3)^2", BF^4~, NO^3-, ethylene, pyridine, etc.) in solutions of such complexes. This may be particularly desirable for complexes with more accessible metal ions, such as planar complexes or complexes having four or fewer coordinating agents, where one or more additional complexing agents could provide additional shielding to the metal from undesirable ligands that might otherwise access the metal and displace a desired complexing agent. [0091] General synthetic chemistry protocols are somewhat different for these classes of molecules due to their propensity to chelate with metallic cations, including copper.

Glassware should be cleaned and silanized prior to use. Plasticware should be chosen specifically to have minimal presence of metal ions. Metal implements such as spatulas should be excluded from any chemistry protocol involving chelators. Water used should be purified by sequential carbon filtering, ion exchange and reverse osmosis to the highest level of purity possible, not by distillation. All organic solvents used should be rigorously purified to exclude any possible traces of metal ion contamination .

[0092] Care must also be taken with purification of such derivatives due to their propensity to chelate with a variety of cations, including copper, which may be present in trace amounts in water, on the surface of glass or plastic vessels. Once again, glassware should be cleaned and silanized prior to use. Plasticware should be chosen specifically to have minimal presence of metal ions. Metal implements such as spatulas should be avoided, and water used should be purified by sequential carbon filtering, ion exchange and reverse osmosis to the highest level of purity possible, and not by distillation . All organic solvents used should be rigorously purified to exclude any possible traces of metal ion contamination . Ion exchange chromatography followed by lyophilization is typically the best way to obtain pure solid materials of these classes of molecules. Ion exchange resins should be washed clean of any possible metal contamination .

[0093] Many of the synthetic routes allow for control of the particular R groups introduced. For synthetic methods incorporating amino acids, synthetic amino acids can be used to incorporate a variety of substituent R groups. The dichloroethane synthetic schemes also allow for the incorporation of a wide variety of R groups by using dichlorinated ethane derivatives. It will be appreciated that many of these synthetic schemes can lead to isomeric forms of the compounds; such isomers can be separated using techniques known in the art.

[0094] Documents describing aspects of these synthetic schemes include the following : ( 1) A W von Hoffman, Berichte 23, 3711 ( 1890); (2) The Polymerization Of

Ethylenimine, Giffin D. Jones, Arne Langsjoen, Sister Mary Marguerite Christine Neumann, Jack Zomlefer, J . Org. Chem., 1944; 9(2); 125-147; (3) The peptide way to macrocyclic bifunctional chelating agents: synthesis of 2-(p-nitrobenzyl)-l,4,7, 10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid and study of its yttrium(III) complex, Min K. Moi et al. , J. Am. Chem. Soc , 1988; 110{18); 6266-6267; (4) Synthesis of a kinetically stable ⁹⁰Y labelled macrocycle-antibody conjugate, Jonathan P L Cox, et al., J. Chem. Soc. Chem. Comm. , 797 (1989) ; (5) Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent, Fritzberg AR, Abrams PG, Beaumier PL, Kasina S, Morgan AC, Rao TN, Reno JM, Sanderson JA, Srinivasan A, Wilbur DS, et al., Proc. Natl. Acad. Sc.i U. S. A. 85(l l) :4025-4029 (1988 Jun); (6) Towards tumour i maging with ¹¹¹In labelled macrocycle-antibody conjugates, Andrew S Craig et al ., J. Chem . Soc. Chem. Comm., 794 ( 1989) ; (7) Synthesis of C- and N-functionalised derivatives of NOTA, DOTA, and DTPA: bifunctional complexing agents for the derivitisation of antibodies, Jonathan P L Cox et al .,, J . Chem. Soc. Perkin . I, 2567 (1990); (8) Macrocyclic chelators as anticancer agents in radioimmunotherapy, N R A Beeley and P R J Ansell, Cu rrent Opinions in Therapeutic Patents, 2 : 1539-1553 (1992) ; and (9) Synthesis of new macrocyclic amino- phosphinic acid complexing agents and their C- and P- functionalised derivatives for protein linkage, Christopher J Broan et al ., Synthesis, 63 (1992) .

[0095] Methods of preparing triethylenetetramines particularly suited for use as described herein are presented in U.S. Patent No. 7582796 (Jonas et al ., issued 1

September 2009), herein incorporated by reference in its entirety. [0096] The triethylenetetramine succinates provided herein are of high purity.

Triethylenetetramine succinates may be produced with purity (calculated on a dry basis) of, for example, at least about 80% triethylenetetramine succinate, at least about 85% triethylenetetramine succinate, at least about 90% triethylenetetramine succinate, at least about 95% triethylenetetramine succinate, at least about 96% triethylenetetramine succinate, at least about 97% triethylenetetramine succinate, at least about 98%

triethylenetetramine succinate, at least about 99% triethylenetetramine succinate, and about 100% triethylenetetramine succinate.

[0097] In addition to the compounds and salt forms provided herein, the invention includes pharmaceutical compositions, including tablets, capsules, solutions, and suspensions for parenteral and oral delivery forms and formulations, comprising a pharmaceutically acceptable carrier and therapeutically effective amounts of one or more of the triethylenetetramine succinates herein provided. Pharmaceutical compositions including the triethylenetetramine disuccinate salts are particularly contemplated, including, for example, pharmaceutical compositions including triethylenetetramine disuccinate an hydrate.

[0098] In human therapy for the treatment of AD, the compounds and their crystal forms described and provided herein, their pharmaceutically acceptable salts, and pharmaceutically acceptable solvates of either entity, can be administered alone, but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. In various examples, they are administered orally in the form of tablets containing pharmaceutically acceptable excipients, such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents. They can also be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration they may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0099] Doses include those previously described (albeit in respect of diseases other than AD) . See Cooper, G.J ., et al ., "Preventing and/or treating cardiovascular disease and/or associated heart failure," U.S. Pat. App. No. 2003/0203973, published October 30, 2003; and Cooper, G.J ., et al ., "Dosage forms and related therapies," PCT Publication No. WO2004/017956, published March 4, 2004. See also U .S. Patent No. 6,897,243, which relates in part to the use of triethylenetetramine in the treatment of diabetes. [00100] For oral, parenteral, buccal and sublingual administration to patients, for example, the daily dosage level of the compounds herein and their pharmaceutically acceptable salts and solvates may be from about 600 mg to about 2400 mg per day (in single doses, although divided doses are specifically contemplated) . Other doses include doses from about 280 mg to about 1680 mg per day, from about 280 mg to about 1400 mg per day, from about 280 mg to about 1120 mg per day per day, from about 560 mg to about 1680 mg per day, from about 560 mg to about 1400 mg per day, from about 560 mg to about 1120 mg per day per day, from about 840 mg to about 1680 mg per day, from about 840 mg to about 1400 mg per day, from about 840 mg to about 1120 mg per day per day.

[OOIOI] Thus, for example, a solid dosage form such as a tablet or capsule may contain from about 200 to about 300 mg, of active compound for administration singly, or two or more at a ti me, as appropriate. In one specifically contemplated embodi ment, a tablet or capsule contains about 280mg triethylenetetramine disuccinate anhydrate. This dosage form is particularly suitable for administration, including self-administration, to a subject suffering from AD. For example, a convenient daily administration of 1 - 4 solid dosage forms, for example 1, 2, 3, or 4 tablets or capsules, comprising for example, about 280 mg triethylenetetramine succinate, such as triethylenetetramine succinate anhydrous, is contemplated to meet an effective daily dosage rate while maxi mizing likelihood of compliance.

[00102] The physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention .

[00103] Generally, in humans, oral administration of the compounds of the invention is the preferred route. In one embodiment, an oral dosing regimen for a typical adult human is from about 560 mg to about 1120 mg per day of triethylenetetramine disuccinate anhydrate when required . Advantageously, oral dosing regimens of from about 560 mg to about 1120 mg per day of triethylenetetramine disuccinate anhydrate, for example, of from 560 mg to about 840 mg per day, can be conveniently met by administration, including self- administration, of 2, 3, or 4 capsules or tablets of about 280 mg triethylenetetramine disuccinate anhydrate. Oral dosage forms of about 280 mg triethylenetetramine disuccinate anhydrate are of a reasonable size, palatable, and capable of ready self-administration by AD sufferers and those at risk of AD, with a view to maximising compliance. Preventative doses are lower, typically from about 1/2 to about 1/10 of the above amounts, including from about 50 mg to about 300 mg per day of triethylenetetramine disuccinate anhydrate. As will be appreciated from the above, such dosage rates can be readily delivered by a single tablet or capsule.

[00104] Thus, the invention provides a pharmaceutical composition comprising a triethylenetetramine succinate provided herein, or a pharmaceutically acceptable solvate thereof, together with a pharmaceutically acceptable diluent or carrier.

[00105] The invention also provides a triethylenetetramine succinate provided herein, or a pharmaceutically acceptable solvate thereof, or a pharmaceutical composition containing any of the foregoing, for use as a human medicament. [00106] In yet another aspect, the invention provides the use of a triethylenetetramine succinate, or a pharmaceutically acceptable solvate thereof, for the manufacture of a human medicament for the curative or prophylactic treatment of AD for which a copper antagonist, particularly a copper (II) antagonist, is indicated .

[00107] In certain embodiments, the pharmaceutical composition or medicament comprising triethylenetetramine succinate, for example, triethylenetetramine disuccinate anhydrate, is a solid oral dosage form.

[00108] A particularly contemplated embodiment of the invention is a solid oral dosage form which contains as the active agent a unit dose of about 200 mg to about 300 mg of triethylenetetramine disuccinate anhydrate. [00109] Typically, a solid oral dosage form as contemplated herein comprises additives conventional in the dosage form in question . Tabletting aids, commonly used in tablet formulation can be used and reference is made to the extensive literature on the subject. These include but are not limited to disintegrants, binders, lubricants, glidants, stabilising agents, fillers or diluents, surfactants and the like. [00110] In various embodiments, contemplated disintegrants include

carboxymethylcellulose (CMC), including CMC-Ca, CMC-Na such as Ac-Di-Sol, and crosslinked CMC, crosslinked PVP (Crospovidone, Polyplasdone of Kollidon XL), alginic acid, radium alginate and guar gu m.

[00111] In various embodiments, contemplated binders include starches, e.g . potato starch, wheat starch, corn starch, microcrystalline cellulose, e.g. products known under the registered trade marks Avicel, Filtrak, Heweten or Pharmacel, hydroxypropyl cellulose, hydroxyethyl cellulose and hydroxypropyl methyl cellulose. [00112] In various embodiments, contemplated glidants include colloidal silica, e.g. Aerosil, magnesium trisilicate, powdered cellulose, starch, talc and tribasic calcium phosphate.

[00113] In various embodiments, contemplated fillers or diluents include confectioner's sugar, compressible sugar, dextrates, dextrin, dextrose, lactose, mannitol, microcellulose, for example Avicel, powdered cellulose, sorbitol, sucrose and talc.

[00114] In various embodiments, contemplated lubricants include Mg, Al or Ca stearate, polyethylene glycol, such as PEG 4000-8000, and talc.

[00115] One or more of these additives can be selected and used by the skilled artisan having regard to the particular desired properties of the solid oral dosage form by routine experimentation and without any undue burden .

[00116] The amount of each type of additive employed, e.g . glidant, binder, disintegrant, filler or diluent and lubricant may vary within ranges conventional in the art. Thus for example, the amount of glidant may vary within a range of from 0.1 to 10% by weight, in particular 0.1 to 5% by weight, e.g . 0.1 to 0.5% by weight; the amount of binder may vary within a range of from about 10 to 45% by weight, e.g. 20 to 30% by weight; the amount of disintegrant may vary within a range of from 2 to 20% by weight, e.g . 15% by weight; the amount of filler or diluent may vary within a range of from 15 to 40% by weight; whereas the amount of lubricant may vary within a range of from 0.1 to 5.0% by weight.

[00117] It is a characteristic of the present solid oral dosage forms that they contain only a relatively small amount of additives given the high content of active agent. This enables the production of physically small unit dosage forms, thereby maximising compliance. The total amount of additives in a given unit dosage may be about 65% or less by weight based on the total weight of the solid oral dosage form, more particularly about 50% or less. In certain embodiments, simple formulations are contemplated, whereby the additive content is below about 45% by weight, for example, below about 35% by weight, including below about 25% by weight or even less.

[00118] The absolute amounts of each additive and the amounts relative to other additives is si milarly dependent on the desired properties of the solid oral dosage form and are chosen by the skilled artisan by routine experimentation without undue burden . For example, the solid oral dosage form may be chosen to exhibit accelerated and/or delayed release of the active agent with or without quantitative control of the release of active agent. Solid dosage forms to provide such delivery kinetics are well known in the art, and can readily be adapted by a person skilled in the art on reading this description .

[00119] Milling, grinding, micronisation, and compaction techniques suitable for use in the preparation of solid dosage forms as herein contemplated are all within the purview of the skilled artisan . For example, triethylenetetramine succinate and any additives present, optionally together with one or more agents effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in a mammalian subject, can be milled either individually or together to suitable particle sizes, for example, particle sizes of from about 0.1 pm to about 200 pm, preferably 1.0 pm to 100 pm . In certain embodiments, at least 90% of the crystals of both the active agent and the additives are present in these ranges. Particles of this size are obtained by conventional comminution methods, e.g. grinding to an air jet mill, hammer and screen mill, fine impact mill, ball mill or vibrator mill .

[00120] Micronisation is preferably effected by known methods using an ultrasonic disintegrator, e.g . of the BRANSON Sonifier type, or by stirring a suspension with an agitator, for example with a stirrer of the HOMOREX type.

[00121] Compaction will typically be carried out using a slugging technique or roller compaction . Roller compaction apparatus is conventional and essentially utilises two rollers which roll towards each other. A hydraulic ram forces one of the rollers against the other to exert a compacting force against the ground particles fed into the roller compactor via a screw conveyor system.

[00122] A compaction force of between 25 and 65 kN is commonly used. Within this range, for each particular formulation the compaction force can be varied in order to obtain a solid oral dosage form wherein the granulate disintegrates into discrete primary particles at a desirable rate. Rapid disintegration can be achieved using minimal compaction forces, while delayed disintegration can be achieved when the solid oral dosage form is compressed above a mini mum compaction force. Rapid disintegration rates enabled using minimal compaction forces is unusual for tablets and can be similar to the disintegration rate of a capsule formulation . The specific minimum compaction force is dependent on the active agent content in any given formulation and on the amount and nature of the additives present.

[00123] The compression of the formulations can be carried out in a conventional tabletting machine, e.g. in an EK-0 Korsch eccentric tabletting machine or a rotary compression machine. The tablets may vary in shape and be, for example, round, oval, oblong, cylindrical or any other suitable shape, and may also vary in size depending on the concentration of the triethylenetetramine succinate and, when present, the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject. A characteristic of the solid dosage forms described herein is their small size having regard to the amount of active agent contained therein, with a view to maximising likelihood of compliance.

[00124] Similar considerations will apply to solid dosage forms comprising capsules or caplets, where the mini misation of additives allows a small physical size per amount of active agent.

[00125] The experiments set forth in the following examples are illustrative of the present inventions and are not intended to li mit the inventions described herein .

EXAMPLES

EXAMPLE 1

[00126] This example investigates metabolic perturbations in various brain regions of AD sufferers. 1. Methods

Acquisition of human brains

[00127] Whole brains from patients and matched controls were obtained from the New Zealand Neurological Foundation Human Brain Bank, in the Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand . All procedures in this study were approved by the University of Auckland Human Participants Ethics Committee with informed consent from all families. The quality of AD brain tissue acquired by the Human Brain Bank was uniformly high and only those with very short postmortem delays (~4-13 h) were used for the current study.

Sampling of human-brain tissue [00128] After receipt into the Human Brain Bank, brains were dissected under the supervision of neuroanatomists, to ensure accurate identification of each of the seven brain regions targeted in this study. The brain regions included three regions known to undergo severe neuronal damage in AD (the hippocampus (HP), entorhinal cortex (ENT) and middle- temporal gyrus (MTG)) three regions known to be less severely affected (the sensory cortex (SCx), motor cortex (MCx) and cingulate gyrus (CG)), and one region known to be relatively spared (the cerebellum (CB)) . Tissue samples of 50 ± 5 mg were dissected from each region and stored at -80 °C until analysis.

Diagnosis and severity of AD

[00129] All AD patients had clinical dementia, whereas controls did not. Control brains were selected from the Human Brain Bank by matching for age, sex and post-mortem delay as shown in Table 1. A consultant neuropathologist diagnosed or excluded AD by applying the Consortium to Establish a Registry for AD (CERAD) criteria.

Table 1: Group characteristics of brains used in study

Variable Control AD

Number 9 9

Age (± SD) 70.1 (±6.7) 70.3 (±7.1)

Male sex, n (%) 5 (55.6) 5 (55.6)

Post-mortem delay (h, range) 9 (5.5-13.0) 7 (4.0-12.0)

Brain weight (g, range) 1260 (1094-1461) 1062* (831-1355)

*P=0.005 compared with Control ; all other differences were non-significant.

Tissue extraction

[00130] Brain tissues placed in "Safe-Lok" microfuge tubes (Eppendorf AG ; Hamburg, Germany) were held at -80 °C until extraction . They then underwent a Folch-style extraction using a TissueLyser batch bead homogeniser (Qiagen ; Manchester, UK) . Briefly, each sample containing 50 ± 5 mg of brain tissue was extracted in 0.8 ml 50: 50 (v/v) methanol : chloroform, to which a solution of the labelled internal standards in methanol had been added to achieve a final concentration of 0.016 mg/ml of each internal standard in the extraction solvent (kept at -20°C until used) . A set of seven isotopically-labelled standards (Citric acid- /4, ¹³C6-D-fructose, Ltryptophan-cfe, L-alanine- /z, stearic acid- /35, benzoic acid- 5, and leucine-c/jo) purchased from Cambridge Isotopes Inc (Tewksbury, MA) were used in this study. Extraction was performed for 10 min at 25 Hz with a single 3-mm tungsten carbide bead per tube. Samples corresponding to the same brain region were handled as single separate batches for this and all subsequent procedures. Separation of phases was achieved by addition of 0.4-ml water followed by vortex-mixing (10-15 s) and centrifugation (2,400 g, 15 min) . After separation, tissue debris lay at the interface between the lower

(non-polar, chloroform) phase and the upper (polar, methanol : water) phase containing the target molecules for the current study. For each batch, extraction blanks were prepared by processing tubes containing solvent and bead, but no tissue sample. This procedure produced clean polar extracts with low levels of lipid and protein content, which are known to otherwise cause response-instability in the GC-MS method.

Sample preparation

[00131] Chloroform in extraction tubes was removed using a 500-μΙ HPLC syringe (Sigma Aldrich, MO, USA) . Tubes were then centrifuged (16,000 g, 15 min) to encourage tissue debris to form a coherent pellet. From the methanol : water supernatant, 200-μΙ aliquots were transferred to pre-labelled tubes containing 600 μΙ of methanol, to precipitate residual protein . A quality-control (QC) pool was made by combining 200-μΙ aliquots from each extraction . The pooled samples were gently mixed and 200-μΙ portions dispensed into tubes containing 600-μΙ methanol . Both sample and QC tubes were centrifuged (16,000 g, 15 min) and 750-μΙ aliquots were transferred to a final set of pre-labelled tubes which were processed to dryness in a Speedvac centrifugal concentrator (~30 °C, 16-18 h (Savant; SPD331 DDA, Thermo Scientific)) . Dried residues were held in sealed tubes at 4 °C for up to one week (shown to be stable for eight weeks for serum previously stored) until

derivatization for GC-MS analysis.

GC-MS Analysis

[00132] Methyloxi me/trimethylsilyl derivatives were prepared by a two-step procedure. GC-MS analysis was performed using an MPS2 autosampler (Gerstel ; Mulhei m an der Ruhr, Germany), a 7890A Gas Chromatograph with Split/Splitless inlet (Agilent; Santa Clara, CA, USA), and a Pegasus HT time-of-flight mass spectrometer (LECO; Stockport, UK).

[00133] Gas chromatography was performed using an Agilent/J&W DB-17MS column (30 m x 0.25 mm x 0.25 pm ; Agilent: # 122-4732) with a 3-m deactivated Fused Silica retention gap (0.25 mm; Agilent: #No 160-2256-10), and helium carrier gas (1.4 ml/min, constant flow mode) . l-μΙ sample injections were made in Pulse Splitless mode at an inlet temperature of 270 °C, using an "empty, hot-needle" technique. The initial column temperature (50 °C) was held for 6 min and then increased to 300 °C at 10 °C /min and held for a further 4 min . This resulted in a total cycle ti me of 42 min between injections. After an initial 450-s solvent delay (to allow solvent and reagents to elute without damaging the detector), mass spectral data were acquired at 10 spectra/s for the range 45-800 Da, detecting a range of amino acids, sugars, sugar alcohols and organic acids as their TMS derivatives. Standard 70-eV electron energy was employed, at a source temperature of 220 °C.

[00134] The study was performed in a series of single-batch experi ments, where each specific brain region constituted a batch. Within each batch, individual cases and controls were randomized, and run in a sequence interleaved with injections of the pooled QC samples (one per four study samples) and extraction blanks (two per batch) . A lead-in sequence of six QC injections at the start of each batch was used to condition the chromatographic system . Extraction blanks were inspected visually to confirm absence of carryover, but not included in subsequent data analysis.

Data reduction

[00135] Data were prepared using the 'Reference Compare' method within ChromaTOF 4.5 (LECO) .

[00136] Briefly, the software was used to perform a global peak deconvolution of parameters to compile a list of nominated 'metabolites', and search mass-spectral libraries to generate putative identities. Databases we employed were : the NIST Mass Spectral Reference Library (NIST08/2008; National Institute of Standards and

Technology/Environmental Protection Agency/National Institutes of Health Spectral Library; NIST, Gaithersburg, MD, USA) ; the Golm Metabolome Database (Max Planck Institute of Molecular Plant Physiology, Potsdam- Golm, Germany); and an in-house library developed at the University of Manchester. Chromatographic retention-ti me data were available from reference standard compounds for a subset of the identities. Within this subset, matching of both mass spectra and expected retention ti me(s) was interpreted to constitute a definitive (D) molecular identification . Matching of mass spectra and retention time with reported data was interpreted as confident (C) identification . Matching of mass spectra only was interpreted as a putative (P) identification . From the list of nominations, we compiled reference tables comprising expected mass spectra and retention-time windows. These were then applied as target lists of features to be searched across all the study samples. As the pooled QC samples should contain all metabolite features encountered in the experiment, these were suitable candidates for compilation of reference tables. To provide a robust reference table, the initial list of nominations was edited to remove ambiguous and low quality spectra prior to application . Global deconvolution was performed on several (3-4) pooled QC injections across the entire experi ment to improve identifications. By displaying these overlaid while editing the list, reproducible spectra were more readily distinguished from lower quality candidates. The same target list was used for all brain regions.

Metabolite abundance reporting

[00137] In order to use the edited reference table as a reporting tool, appropriate parameters such as mass spectral match thresholds and tolerable retention-time deviations (6 sec) were specified, and the table initialized using a pooled QC sample to provide reference m/z peak areas. Improved reproducibility was achieved by the use of internal standard ratios rather than raw peak areas.

[00138] The most suitable standard was assigned to each metabolite by determining which internal standard yielded the lowest variance for a given metabolite across all the QC injections.

[00139] The resulting data for each experi ment were compiled into a matrix of metabolite-intensity data, which was merged with experimental metadata for visualization and statistical analysis. Although the automated procedure was highly reliable (esti mated return of correct peak areas for >95% of features measured), data sets were also curated manually to remove possible integration errors which were mostly associated with metabolites showing non-ideal peak shape.

Statistics

[00140] The merged metadata were used for data analysis. A principal-components analysis (PCA) was performed for visualization to confirm overall data integrity, using SIMCA-P software (UMetrics AB, Umea, Sweden) . Calculation of relative fold-change and statistical analysis were performed in log space using multiple t-tests (GraphPad Prism 6) . Data were considered for multiple comparison analysis applying an FDR ( 10%) correction . The fold-changes were converted to linear space for presentation and metabolites identified in≥ 5 samples in each group were reported . 2. Results

[00141] There was about 16% median brain weight decline in AD; median (range) brain weight was 1,062 g (831-1,355) in AD and 1,260 g (1,094-1,461 ; P<0.005) in controls (see Table 1) .

[00142] PCA of GC-MS data revealed : 1) excellent class separation in all brain regions between AD and control samples; 2) greater biological than technical variation ; and 3) absence of run-order effects in this study (data not shown) . One control sample clustered more closely to the AD samples. The brain from which this sample originated had the lowest brain-weight (1,094 g) among the controls and was assessed as Braak stage II by neuropathological examination . On this basis, this control sample was reclassified as a case of preclinical AD case and has been excluded from the subsequent analysis for reasons of clarity.

[00143] 69 metabolite features were categorised per brain region . The metabolites for which an altered abundance in one or more regions of the AD was observed are shown in Table 2. Numbers indicate fold-change (AD/controls) . Changes with P<0.05 (10% FDR) were considered significant and were shown in bold italic font. 55 features were shown to change in at least one brain region (FDR corrected multiple t-test (p < 0.05)), with individual regions showing between 16 and 33 metabolites identified as significantly changed . [00144] Metabolites for which no altered abundance was observed for any brain region are shown in Table 3.

Table 2: Relative fold change in metabolites with altered abundance in the AD brain.

Metabolite HP ENT MTG sex MCX CG CB

Glucose and related metabolites & pentose phosphate pathway components

Glucose (D) 12.3 9.3 16.9 10.8 8.0 9.9 6.8

Glucose-6-phosphate (D) 5.9 4.9 8.5 4.8 6.0 4.8 3.8

Sorbitol (D) 3.4 3.8 3.9 5.0 5.3 5.0 4.6

Fructose (D) 4.6 4.8 7.0 6.8 7.0 7.0 7.1

Fructose-6-phosphate (D) 1.2 0.6 5.3 1.6 2.5 7.7 2.3

Pentonic acid A (P) 1.1 1.3 1.3 1.2 1.4 1.4 1.3

Pentonic acid B (P) 2.1 2.0 1.9 1.7 1.7 1.9 1.7

Arabinose (P) 3.5 NM 3.6 3.5 6.8 3.9 NM

Ribose-5-phosphate (D) 0.8 0.6 0.8 1.1 1.1 1.0 0.9

Erythronic acid (P) 1.6 1.8 1.1 1.2 1.2 1.4 1.5

Alternative fuel source

Butanediol (D) 4.2 4.3 1.6 4.2 9.3 4.1 4.1 β-Hydroxybutyric acid (D) 1.6 2.5 1.5 3.4 1.8 2.5 1.6

Lactic acid (D) 2.6 0.5 1.3 3.4 7.3 1.7 0.6

2-hydroxy-3-methylbutyric acid (P) 7.7 2.3 2.8 4.3 2.5 4.7 3.4

Threitol (D) 2.0 2.3 1.8 2.4 2.3 2.4 3.2

Xylitol (D) 1.7 1.3 1.2 1.2 1.1 1.4 1.7

Disaccharide (D) 4.8 2.3 3.2 3.6 1.8 1.8 0.9

N-acetylglucosamine (C) 1.2 1.1 1.7 1.4 1.4 1.6 2.8 myo-Inositol (D) 1.1 1.9 1.1 1.2 0.8 0.7 0.9 myo-Inositol-l-phosphate (P) 2.3 2.0 4.1 1.7 2.2 3.9 3.5

Glycerol (D) 0.6 0.7 0.8 0.8 0.7 0.9 0.9

Glycerol-2-phosphate (P) 1.9 1.6 1.5 1.7 1.8 1.6 1.6

Glycerol-3-phosphate (D) 2.3 2.7 1.4 2.5 2.6 1.8 1.6

Glyceric acid (P) 1.3 1.1 2.8 1.5 1.1 2.6 1.8 TCA cycle & urea cycle

Citric acid (D) 1.7 2.1 1.7 1.9 1.1 1.1 1.1

Malic acid (C) 1.6 1.9 2.4 1 0.8 1.7 0.9

Fumaric acid (C) 1.8 1.3 1.7 1.2 0.8 1.4 1.3

Ornithine (D) 0.6 0.6 1.0 0.7 0.7 0.9 0.3

Urea (D) 6.5 5.6 4.7 4.9 5.0 5.3 4.9

N-acetylglutamic acid (D) 0.4 0.8 0.9 0.8 0.7 0.8 1.0

Creatinine (D) 1.1 1.0 1.5 1.0 1.0 1.2 1.2

Amino acids

Proline (D) 0.5 0.4 0.5 0.7 0.8 0.8 0.5

Lysine (D) 0.5 0.7 1.1 0.8 1.1 1.1 0.3

Glycine (D) 0.9 0.7 0.8 0.8 0.7 0.9 0.9

Serine (D) 0.7 0.6 1.0 0.7 0.8 1.0 0.7

Threonine (D) 0.8 1.3 1.5 1.1 2.2 1.4 1.7

Cysteine (D) 2.0 1.4 0.7 1.0 1.0 1.4 0.9 beta-Alanine (D) 1.2 1.0 1.3 1.1 1.1 1.3 0.9

Aspartic acid (D) 0.6 0.6 0.8 1.0 0.9 0.9 0.8

N-acetylaspartic acid (D) 0.7 0.8 0.7 1.0 0.9 0.9 1.0

Glutamic acid (P) 1.3 1.0 1.0 1.3 1.3 1.3 2.6

GABA (D) 1.3 0.6 0.5 0.7 0.7 0.9 0.8

4-hydroxybutyric acid (C) 0.6 0.6 1.1 0.8 0.8 0.7 0.7

Phenylalanine (D) 1.3 1.2 2.1 1.6 1.8 2.0 1.3

Tryptophan (D) 2.5 2.2 4.7 1.8 3.2 4.0 1.1

Nucleosides

Adenine (D) 1.0 1.0 0.9 1.0 0.9 0.9 1.7

Uracil (C) 0.6 0.5 0.6 0.6 0.5 0.6 0.7

Adenosine-5-monophosphate (P) 1.6 2.1 1.9 1.4 NM 2.4 1.3

Guanosine (D) 0.7 0.8 2.8 0.9 NM 2.5 1.6

Hypoxanthine (D) 0.7 0.7 0.7 0.7 0.7 0.8 0.7

Miscellaneous

Ethanolamine (D) 0.6 0.4 0.5 0.5 0.5 0.6 0.6

Methyl-phosphate (C) 0.7 0.4 0.4 0.7 0.6 0.5 0.8

Phosphoric acid (D) 1.2 1.1 0.5 0.8 0.7 0.6 0.9

2-Hydroxyglutaric acid (D) 1.9 2.2 1.9 1.6 1.6 1.8 1.4

Ascorbic acid (P) 2.0 1.7 1.1 1.8 2.2 1.6 1.5

Abbreviations: D, definitive; C, confident; P, putative. Changed with P<0.05 were considered significant and are shown in bold italic font. Table 3: Relative fold change in metabolites with no statistically significant change in abundance in the AD brain.

Metabolite HP ENT MTG sex MCX CG CB

Hydroxylamine (P) 1.0 1.1 0.8 1.0 1.2 0.9 1.2

Alanine (D) 0.8 0.7 1.3 0.6 3.4 1.0 1.2

Pyruvic acid (D) 1.3 1.6 1.5 0.8 0.5 1.0 0.8

Valine (D) 1.6 0.9 2.8 1.0 1.0 1.5 0.5

Leucine (D) 0.9 0.9 1.4 1.0 1.0 1.4 0.8

Isoleucine (D) 0.8 0.8 1.3 0.8 1.0 0.7 1.0

Succinic acid (C) 1.3 0.6 1.1 1.2 1.3 1.2 1.2

Methionine (D) 1.0 0.9 1.3 0.9 1.0 1.4 0.7

Ribotol (P) 0.3 1.0 2.0 0.4 1.2 1.1 1.0

Pyroglutamic acid (D) 1.2 1.2 1.1 1.3 1.5 1.2 1.1

Mannitol (D) 1.1 1.2 1.3 1.3 1.2 1.2 1.8

Scyllo-inositol (D) 0.9 0.9 1.1 0.7 0.7 0.8 0.7

Tyrosine (D) 0.7 0.7 1.2 1.0 1.0 1.2 0.6

Adenosine (D) 2.0 1.3 0.9 1.0 1.0 0.8 1.0

Abbreviations: D, definitive; C, confident; P, putative.

[00145] This example demonstrates alterations in metabolites from several pathways involved in glucose clearance and utilisation.

EXAMPLE 2

[00146] This example investigates copper metal levels in various brain regions of AD sufferers.

1. Methods

[00147] Human brains were acquired and sampled as for Example 1. Study group characteristics are shown in Table 4.

[00148] The diagnosis and severity of AD was assessed as for Example 1. Table 4: Group characteristics of brains used in study

Variable Control AD

Number 13 9 Age (± SD) 70 (±6.3) 70.3 (±7.1)

Male sex, n (%) 7 (53.8) 5 (55.6)

Post-mortem delay (h, range) 12 (5.5-15.0) 7* (4.0-12.0)

Brain weight (g, range) 1260 (1094-1461) 1062* (831-1355)

Wet-wt/dry-wt 5.7 (5.6-5.9) 5.5 (5.4-5.6)

Plaques, n (%) 1 (7.7) 9 (100)**

Tangles, n (%) 1 (7.7) 9 (100)**

*P=0.005, **P<0.0001 compared with Control ; all other differences were non-significant.

Preparation of brain extracts

[00149] Cu concentrations were determined on a dry-weight basis. Brain samples of 50 ± 5 mg wet-weight were first dried to constant weight in a centrifugal concentrator (Savant Speedvac; Thermo-Fisher, Waltham, MA) . The precise weights of the dried tissue samples were measured to enable normalization of tissue-metal content by dry-weight (Table 1) . Samples were digested in 2-mL microcentrifuge tubes (eppendorf) as described below. Digestion

[00150] Tissue was digested using concentrated nitric acid (A509 Trace Metal Grade; Fisher, Loughborough, UK) to which was added 5% (v/v) Agilent Internal Standard mixture (5183-4681 ; Agilent Technologies, Cheadle, UK) . This internally-standardized acid was also used at appropriate dilutions to provide rinse and calibration solutions, at 2% (v/v) final nitric acid concentration .

[00151] Calibration solutions were produced by appropriate dilutions of Environmental Calibration Standard (Agilent 5183-4688) . Acid digestion was carried out using an Open- vessel' method. Tissue aliquots were briefly centrifuged to ensure that the tissue sat at the bottom of the tube. The tube lids were punctured to prevent pressure build-up, and 0.2 ml_ standard-containing nitric acid added. Tubes were then inserted into a "Dri-block" heater which was initially at room temperature. Tubes with standard-containing acid but no sample were processed in each batch to provide "digestion" blanks. Temperature was then set to 60oC and the block switched on . After 30 min, the set temperature was increased to lOOoC, and digestion continued for a further 210 min . After digestion, the tubes were allowed to cool overnight.

[00152] Aliquots of 100 μΙ_ were taken from each digestion and added to 15-mL Falcon tubes (Greiner) containing 5 ml_ LC-MS grade water, to produce solutions for analysis at a final nitric acid concentration of 2% (v/v) . ICP-MS

[00153] Cu-metal concentrations were measured using an Agilent 7700x ICP-MS spectrometer equipped with a MicroMist nebulizer (Glass Expansion, Melbourne, Australia) and a Scott double-pass spray chamber. Nickel sample and skimmer cones were used. Sample introduction was performed using an Agilent Integrated autosampler (I-AS). Helium was used as the collision gas. A multi-element method including all elements present in the calibration solution was applied. Calibration solutions were produced by appropriate dilutions of Environmental Calibration Standard (Agilent 5183-4688). Scandium was used as the internal standard. Two collision cell gas modes were applied, Cu metal concentration was analysed in helium mode (5.0 mL. min-1 helium). Mode selection followed Agilent recommendations to minimize interference for measured elements by e.g. isobaric cluster ions. Integration time was 0.3 s. For each analytical batch, multi-element calibration was performed using serial dilutions of the calibration standard. An intermediate concentration from this calibration series was used as a periodic quality-control (QC) sample throughout each analytical batch. Instrument and digestion blanks were also interspersed through each set of randomized samples. The detection limit for Cu was determined by comparison of calibration samples and blanks and any samples below this level were eliminated prior to reporting.

[00154] ICP-MS measures the amount of metals such as copper in their elemental state. In contrast, most metals are present in the human body as cations; in the case of copper, the physiological cations copper(I) and copper(II).

Data analysis

[00155] Datasets were exported to Microsoft Excel worksheets and individual values of each sample were normalized by the corresponding sample weight (dry weight). Weight- adjusted datasets were then log-transformed for statistical analysis. Means (± 95% CI) of the log-transformed data were calculated and the significance of between-group differences was examined by unpaired t-test with Welch's correction to allow for unequal variances and sample sizes. Means (± 95% CI) were back-transformed to reflect the actual elemental concentrations of elements. Statistical calculations were performed using GraphPad v6.04 (Prism; La Jolla, CA). P-values of <0.05 have been considered significant, and those of 0.05 < P < 0.10 have also been tabulated.

2. Results

[00156] The concentration of the essential metal Cu was measured in seven regions of human post-mortem brains from nine AD and 13 control subjects matched for age and sex. [00157] One control patient also had neuropathological findings consistent with AD (Braak Stage II) and was therefore diagnosed with premanifest disease : this finding is consistent with the known frequency of asymptomatic AD in similarly-aged groups in the study population . Wet-wt/dry-wt ratios did not differ significantly between cases or controls.

[00158] Mean Cu concentrations in control tissue were highest in CB (710 pmol/kg dry-wt) and lowest in ENT (310 mol/kg dry-wt) as shown in Table 5. Cu levels were significantly decreased compared with corresponding regional -control values in all seven regions of AD-brain, consistent with the presence of pan-cerebral copper deficiency. Table 5: Copper metal concentration and wet/wt/dry-wt ratios in seven brain regions of AD and control brains.

Cu (pmol/kg dry-wt, mean ± 95% CI)

Brain region Control AD p-value

HP 330 (260-419) 183 ( 128-261) 0.0066

ENT 309 (281-340) 202 ( 146-281) 0.018

MTG 356 (300-422) 239 ( 195-293) 0.0035

sex 382 (323-451) 268 (218-330) 0.0079

MCX 390 (319-478) 252 ( 184-347) 0.019

CG 346 (264-454) 198 ( 141-280) 0.010

CB 708 (617-811) 374 (323-434) < 0.0001

Reference isotope = ⁵³Cu . Data are means (± 95% CI) ; P-values for significance of between-group differences were calculated by Welch's modified t-tests based on

measurements from control (n = 13) and AD (n=9) brains.

[00159] This example demonstrates pan-cerebral copper-deficiency in seven regions of the AD brain .

EXAMPLE 3

[00160] This example investigates levels of metabolites and trace metals in brain regions known to undergo varying degrees of damage in AD.

1. Methods

[00161] Human brains were acquired and sampled as for Example 1. The study group was the same as that for Example 1. No patients had history consistent with type II diabetes. [00162] The diagnosis and seventy of AD was assessed as for Example 1.

Analytical methods

[00163] Metabolite levels were compared between cases and controls by GC-MS-based metabolomics in wet-tissue. Copper levels were measured in dry-tissue by inductively- coupled-plasma mass spectrometry (ICP-MS) .

Observational case-control study

[00164] Patients and controls without cognitive impairment were respectively recruited through memory clinics in Leeds and Dewsbury (England), and the Leeds Family Health Services Authority day hospitals and elderly medicine outpatient clinics in the Leeds area. All were of European Caucasian background and gave written informed consent (consent from relatives of the AD-patients was provided, where appropriate) . Diagnosis of probable AD was made in accordance with international diagnostic criteria (National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association Work Group : NINCDS-ADRDA) . All participants underwent a standardised clinical evaluation : medical history, fasting plasma glucose (FPG) and HbAlc, and cognitive function assessment by Mini-Mental State Examination (MMSE) .

[00165] AD samples were selected by excluding patients with diagnosed T1D or T2D including those on synthetic insulin . Patients with a previous medical history of other medical conditions were also excluded. Samples were then selected from the whole-study population for whom required measurements (FPG, HbAlc) were available. The resulting 42 AD patients were then age- and gender-matched to 43 controls.

2. Results

Glucose, sorbitol and fructose in AD brain regions

[00166] The relative fold-changes in glucose, sorbitol and fructose in seven brain regions is shown in Table 6.

[00167] Levels of glucose were elevated in all brain regions of AD patients. Levels of glucose tended to be higher in regions of the brain more severely affected by AD such as the middle temporal gyrus.

[00168] Sorbitol, formed from glucose, is the first metabolite in the polyol pathway, which usually accounts for a few percent at most of glucose utilization under normal conditions. Sorbitol was elevated in all AD brain regions. [00169] Fructose is the second metabolite in the polyol pathway. Brain fructose levels were elevated in all AD-brain regions.

Table 6: Relative fold change in glucose, sorbitol and fructose in brain regions.

Lower Upper P- BH FDR

Metabolite Brain region Estimate

Bound Bound value Q-value

Glucose Cerebellum 5.2 1.4 18.6 0.015 0.015

Entorhinal cortex 7.4 2.4 23.2 0.0028 0.0041

Cingulate gyrus 8.0 2.4 26.1 0.0023 0.0037

Hippocampus 8.9 2.9 27.4 0.0016 0.0030

Sensory cortex 9.2 3.0 28.8 0.0014 0.0029

Motor cortex 11.8 3.7 37.7 0.0006 0.0019

Middle temporal gyrus 16.4 5.2 51.2 0.0002 0.0019

Sorbitol Cerebellum 3.7 1.6 8.6 0.0036 0.0044

Entorhinal cortex 4.3 2.0 9.1 0.0006 0.0019

Cingulate gyrus 3.1 1.5 6.5 0.0048 0.0053

Hippocampus 4.1 1.9 8.5 0.0009 0.0021

Sensory cortex 4.1 1.9 8.6 0.0009 0.0021

Motor cortex 3.0 1.4 6.6 0.0090 0.0094

Middle temporal gyrus 3.3 1.5 6.9 0.0039 0.0046

Fructose Cerebellum 5.3 2.0 14.4 0.0018 0.0031

Entorhinal cortex 5.7 2.4 13.5 0.0004 0.0019

Cingulate gyrus 3.9 1.7 9.2 0.0030 0.0041

Hippocampus 5.5 2.3 12.9 0.0005 0.0019

Sensory cortex 5.4 2.4 12.4 0.0004 0.0019

Motor cortex 4.2 1.7 10.2 0.0032 0.0041

Middle temporal gyrus 5.7 2.6 12.9 0.0003 0.0019

Estimates and their lower and upper bounds were derived by Bayesian modelling. Abbreviation : 'BH FDR Q-value' is the Benjamini-Hochberg False-Discovery Rate- adjusted P-value. [00170] Elevated brain levels of glucose, sorbitol and fructose were present in one control patient, a 76 year-old female, who had no ante-mortem clinical evidence for brain disease or dementia, but had premanifest AD characterised by low brain weight (1,094 g) and positive post-mortem histology. Copper levels in AD brain regions

[00171] Mean (±95% CI) brain-copper levels summed across all seven regions were 256 (232-279) μΓηοΙ/dry kg in AD and 406 (363-449) pmol/dry kg in controls (P= 1.6xlO^"8) : there was thus an overall decrease of ~40% in brain-copper levels in AD. Mean brain-copper levels tended to be lower in AD than controls in all brain regions examined as shown in Figure I B. In addition, there was moderate evidence for a trend in copper levels to be inversely proportional to tissue-glucose values in AD-patients (P=0.021 ; restricted iterative generalized least squares, RIGLS 23) but not in controls as shown in Figure 1 (A and B) .

Plasma glucose and plasma copper levels in AD patients [00172] In the case control study group, measured cognitive function (MMSE score) in the AD group was significantly lower than that of controls whereas the ApoE4 allele was more prevalent (data not shown) . Fasting plasma glucose (FPG) and serum HbAlc levels were equivalent between groups so there was no evidence for elevated rates of undiagnosed T2D or impaired glucose tolerance (IGT)/i mpaired fasting glucose (IFG) in this group of British patients with sporadic AD (data not shown) . FPG and fasting plasma-copper levels did not differ significantly between the AD group and the controls (data not shown) .

[00173] This example demonstrates elevated glucose, fructose, sorbitol and copper levels in the AD brain .

INDUSTRIAL APPLICABILITY [00174] The present invention finds application in the medical and pharmaceutical fields, providing methods and compositions for the treatment Alzheimer's disease, for i mproving physiological and/or neurological deficits associated with Alzheimer's disease.

Claims

1. A method of treating Alzheimer's disease (AD) by administration to a mammalian subject in need thereof an effective amount of a triethyienetetramine succinate.

2. The method of claim 1 wherein the triethyienetetramine succinate binds Cu(II).

3. The method of claim 2 wherein the triethyienetetramine succinate is specific for Cu(II) over Cu(I) .

4. The method of claim 3 wherein the triethyienetetramine is selected from the group comprising triethyienetetramine (2,2,2 tetramine), 2,3,2 tetramine, 3,3,3 tetramine, and salts, active metabolites, derivatives, and prodrugs thereof.

5. The method of claim 4 wherein the triethyienetetramine succinate is

triethyienetetramine disuccinate anhydrous.

6. The method of claim 1 wherein the triethyienetetramine succinate is a polymorph of triethyienetetramine succinate having a differential scanning calori metry (DSC) extrapolated onset/peak melting temperature of from between about 170°C to about 190°C.

7. The method of claim 1 wherein the triethyienetetramine succinate is a crystalline triethyienetetramine succinate in the form of a crystal having alternating layers of triethyienetetramine molecules and succinate molecules.

8. The method of claim 1 wherein the method comprises administering to a mammalian subject in need thereof one or more of a dosage form comprising from about 250 mg to about 280mg of triethyienetetramine disuccinate anhydrate.

9. The method of claim 8 wherein the method comprises administering to a mammalian subject in need thereof one or more of a dosage form comprising about 280mg of triethyienetetramine disuccinate anhydrate.

10. The method of claim 1 wherein the method additionally comprises administration to the subject of an effective amount of an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject.

11. The method of claim 10 wherein the agent effective to reduce the amount or

concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is selected from the group comprising amylin, an amylin analogue, a GLP-1 agonist, and a selective dipeptidyl peptidase (DPP-IV) inhibitor.

12. The method of claim 11 wherein the amylin analogue is Symlin .

13. The method of claim 11 wherein the GLP-1 agonist is exenatide, liraglutide,

lixisenatide, albiglutide, or dulaglutide, or any combination of two or more thereof.

14. The method of claim 11 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Gemigliptin, Dutogliptin, and

Omarigliptin .

15. The method of claim 11 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising alogliptin, linagliptin, saxagliptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia.

16. The method of any one of clai ms 10 to 15 wherein the administration of the

triethylenetetramine succinate and of the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is simultaneous, sequential, or separate.

17. The method of any one of clai ms 1 to 16 wherein the mammalian subject is a non- diabetic subject or a subject who is not undergoing treatment for diabetes.

18. The method of any one of clai ms 1 to 17 wherein the mammalian subject does not suffer from Wilson's disease.

19. The method of any one of clai ms 1 to 18 wherein the mammalian subject is a human subject.

20. A pharmaceutical composition comprising triethylenetetramine succinate anhydrous.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is a solid dosage form formulated for oral administration .

22. The pharmaceutical composition of claim 20 or claim 21, wherein the pharmaceutical composition comprises from about 200 to about 300 mg triethylenetetramine succinate anhydrous and is formulated for oral administration.

23. A pharmaceutical composition comprising triethylenetetramine succinate anhydrous, additionally comprising or formulated to be administered in conjunction in with an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in a mammalian subject, wherein the one or more pharmaceutical compositions comprises a pharmaceutically acceptable carrier or diluent.

24. The pharmaceutical composition of claim 23 wherein the pharmaceutical composition is a solid dosage form comprising from about 200 mg to about 300 mg

triethylenetetramine succinate anhydrous and is formulated for oral administration .

25. The pharmaceutical composition of any one of clai ms 20 to 30 wherein the solid

dosage form is a tablet or capsule comprising about 280 mg triethylenetetramine succinate anhydrous.

26. The pharmaceutical composition of any one of claims 23 to 25 wherein the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is selected from the group comprising amylin, an amylin analogue, a GLP-1 agonist, and a selective dipeptidyl peptidase (DPP-IV) inhibitor.

27. The pharmaceutical composition of claim 26 wherein the amylin analogue is Symlin .

28. The pharmaceutical composition of claim 26 wherein the GLP-1 agonist is exenatide, liraglutide, lixisenatide, albiglutide, or dulaglutide, or any combination of two or more thereof.

29. The pharmaceutical composition of claim 26 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin .

30. The pharmaceutical composition of claim 26 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising alogliptin, linagliptin, saxagliptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia.

31. The pharmaceutical composition of claim 23 comprising triethylenetetramine

disuccinate and amylin or Symlin .

32. The pharmaceutical composition of claim 23 comprising triethylenetetramine

disuccinate and one or more agents selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin .

33. The pharmaceutical composition of claim 23 comprising triethylenetetramine

disuccinate and one or more agents selected from the group comprising alogliptin, linagliptin, saxagliptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia.

34. A method of treating AD in a mammalian subject in need thereof or suspected of being in need thereof, the method comprising : analysing an encephalic sample from said subject to determine the amount or concentration of glucose, fructose, or sorbitol in the sample, and comparing the amount or concentration of glucose, fructose, or sorbitol in the sample to the amount or concentration of glucose, fructose, or sorbitol found in a mammalian subject of the same species not suffering from AD, wherein an elevated amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose in the sample is indicative of an increased risk of developing AD or of the presence of AD, and administering to a mammalian subject determined to be at an increased risk of developing AD or suffering from AD an effective amount of a triethylenetetramine succinate.

35. A method of assessing a mammalian subject's risk of developing AD which comprises: analysing an encephalic sample from said subject to determine the amount or concentration of glucose, fructose, or sorbitol in the sample, and comparing the amount or concentration of glucose, fructose, or sorbitol in the sample to the amount or concentration of glucose, fructose, or sorbitol found in a mammalian subject of the same species not suffering from AD, wherein an elevated amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose in the sample is indicative of an increased risk of developing AD, and optionally administering to the mammalian subject an effective amount of a triethylenetetramine succinate.