Improved cognitive ability

Abstract

The present invention relates to acetylglucosamine. Specifically, the invention relates to an application of acetylglucosamine in improving cognitive competence.

Description

Technical Field

[0001] The present invention relates to acetylglucosamine. Specifically, the present invention relates to the use of acetylglucosamine in improving cognitive ability. However, it should be understood that the present invention is not limited to this specific field of use.

[0002] Related Applications

[0003] This application claims priority to Australian provisional application 2023902258 (filing date July 14, 2023), the contents of which are hereby incorporated by reference in their entirety. Background Art

[0004] Cognitive impairment, or disturbances in memory and learning processes, has a significant adverse impact on quality of life – both for the person with the condition and for those who care for them. Clinically recognised cognitive impairment ranges from mild cognitive impairment to dementia of varying severity. Cognitive impairment may also be associated with a variety of diseases or disorders, such as schizophrenia, depression or Parkinson's disease.

[0005] Dementia is a clinically recognized broad spectrum syndrome resulting in a progressive loss of cognitive abilities. Dementia may be one of many symptoms of various neurological diseases or may be the primary abnormality associated with the disease, as is the case with Alzheimer's disease (AD). Common causes of dementia include brain atrophy associated with AD, Lewy body disease (or dementia with Lewy bodies), frontotemporal lobar disease, Pick's disease, narrowing or blockage of cerebral blood vessels (i.e., vascular dementia, also called multi-infarct dementia), Huntington's disease, Parkinson's disease, head trauma, HIV infection, or Down syndrome.

[0006] AD is the most common cause of dementia in older adults. AD is a chronic neurodegenerative disease that was previously understood to be a variant of Alzheimer's disease. AD affects parts of the brain involved in thinking, memory, and language. Therefore, the most common early symptom is often difficulty remembering recent events (short-term memory loss). As the disease progresses, AD may show symptoms such as language problems, disorientation (including getting lost easily), mood swings, loss of motivation, decreased ability to take care of oneself, and behavioral problems. Mild cognitive impairment (MCI) is considered a transitional stage between the cognitive changes of normal aging and the more serious problems caused by AD. People with MCI have less severe symptoms than those with AD and are usually able to be self-sufficient and carry out normal daily activities.

[0007] It is estimated that 4 million people in the United States may currently have AD. Symptoms of AD usually appear between the ages of 65 and 90. Although the risk of AD increases with age, AD is not part of the normal aging process. While younger people may also have AD, it is less common. About 3% of men and women aged 65 to 74 have AD, and nearly half of people over the age of 85 may have the disease.

[0008] At present, the etiology of AD is still controversial, and a variety of disease pathological mechanisms have been proposed. However, one of the hallmarks of AD is the accumulation of amyloid plaques in the brain. Amyloid plaques are extracellular deposits of aggregates formed by amyloid β (Aβ) peptides. Another characteristic of AD is the presence of neurofibrillary tangles, which are intracellular aggregates of hyperphosphorylated tubulin-associated unit proteins (Tau). These amyloid plaques and neurofibrillary tangles have been considered as histopathological hallmarks of AD.

[0009] There is no cure for AD and related dementias. Furthermore, only a few drugs have been shown to provide at most modest and mostly transient benefits to patients with cognitive impairment. These drugs not only have limited efficacy, but also have significant side effects and high costs, leading to treatment discontinuation in some cases. As life expectancy increases and the aging of the population intensifies, there is a need to develop treatments that delay or mitigate cognitive decline in elderly patients or improve cognitive function in individuals with AD or related dementias.

[0010] The reference to any prior art in the specification does not constitute an acknowledgement or implication that the prior art forms part of the common general knowledge in any jurisdiction, nor does it imply that a person skilled in the art could reasonably expect that the prior art could be understood, regarded as relevant and / or combined with other prior art. Summary of the invention

[0011] The present inventors have unexpectedly discovered that cognitive impairment can be treated by administering acetylglucosamine.

[0012] According to a first aspect, the present invention provides a method for treating cognitive impairment in a subject, comprising administering acetylglucosamine to the subject. The present invention also provides a method for treating a preclinical cognitive condition in a subject, comprising administering acetylglucosamine to the subject. Optionally, the subject is identified as being at risk for cognitive impairment. The present invention also provides a method for preventing cognitive impairment or a preclinical cognitive condition in a subject, comprising administering acetylglucosamine to the subject. Optionally, the subject is identified as being at risk for cognitive impairment or a preclinical cognitive condition.

[0013] According to a second aspect, the present invention provides the use of acetylglucosamine in the manufacture of a medicament for treating cognitive impairment in a subject. The present invention also provides the use of acetylglucosamine in the manufacture of a medicament for treating a preclinical cognitive condition in a subject. Optionally, the subject is identified as being at risk for cognitive impairment. The present invention also provides the use of acetylglucosamine in the manufacture of a medicament for preventing cognitive impairment or a preclinical cognitive condition in a subject. Optionally, the subject is identified as being at risk for cognitive impairment or a preclinical cognitive condition.

[0014] According to a third aspect, the present invention provides a composition comprising acetylglucosamine for use in treating cognitive impairment in a subject. The present invention provides a composition comprising acetylglucosamine for use in treating cognitive impairment in a subject.

[0015] In one embodiment, acetylglucosamine has the following structure:

[0016]

[0017] wherein at least one of R1, R2, R3 and R4 is an acetyl group, and the others are independently H or an acetyl group,

[0018] or a pharmaceutically acceptable salt thereof.

[0019] In one embodiment, R1, R2, R3 and R4 are all acetyl groups. In one embodiment, two of R1, R2, R3 and R4 are acetyl groups. In one embodiment, three of R1, R2, R3 and R4 are acetyl groups. Combinations of acetylglucosamine of

[0014] and

[0015] are also contemplated. Optionally, one, two, three or four of R1, R2, R3 and R4 are all acetyl groups.

[0020] In one embodiment, acetylglucosamine is administered to a subject by a route selected from the group consisting of oral, nasal, buccal, sublingual, intravenous, transmucosal, parenteral, by inhalation, spray, transdermal, subcutaneous, intrathecal, topical, and rectal.

[0021] In one embodiment, acetylglucosamine is administered orally to the subject. Alternatively, oral administration is oral administration of a composition comprising acetylglucosamine and one or more pharmaceutically acceptable excipients. Preferably, the composition is solid, more preferably in the form of tablets, capsules or powders. Pharmaceutically acceptable excipients are optionally fillers, solid carriers, coating agents, diluents, lubricants, adhesives, taste masking agents, preservatives, antibacterial and antifungal agents, isotonic agents and absorption delay agents, any physiological buffer (i.e., pH value is about 7.0 to 7.4) medium, including suitable water-soluble carriers, conventional solvents, dispersion media. Other conventional additives include lactose, mannitol, corn starch, potato starch, adhesives (e.g., crystalline cellulose, cellulose derivatives, gum arabic, gelatin), disintegrants (e.g., sodium carboxymethyl cellulose) and lubricants (e.g., talc or magnesium stearate).

[0022] In one embodiment, acetylglucosamine reduces the expression of exosteoin glycosyltransferase 1 (EXT1) in the brain of a subject.

[0023] In one embodiment, acetylglucosamine reduces the expression of UDP N-acetylglucosamine in the brain of a subject.

[0024] In one embodiment, acetylglucosamine reduces the level of hypersulfated heparan sulfate in the brain of a subject.

[0025] In one embodiment, the cognitive disorder is Alzheimer's disease (AD).

[0026] In one embodiment, AD is early-onset AD.

[0027] In one embodiment, AD is late-onset AD.

[0028] In one embodiment, the AD is an UppsalaAPP deletion form of AD.

[0029] In one embodiment, acetylglucosamine increases the levels of glucosamine-6-phosphate and glucose-6-phosphate in the brain of a subject. Preferably, acetylglucosamine increases the level of glucosamine-6-phosphate in the brain of a subject compared to the level of glucosamine-6-phosphate in the brain of a subject before administration of acetylglucosamine.

[0030] In one embodiment, acetylglucosamine increases the level of fructose-6-phosphate in the brain of a subject.

[0031] In one embodiment, acetylglucosamine reduces seizure-related mortality in a subject. In one embodiment, acetylglucosamine reduces the frequency of seizure-related mortality in a population of subjects.

[0032] In one embodiment, acetylglucosamine reduces spatial working memory deficits in a subject.

[0033] As used herein, unless the context requires otherwise, the term "comprises" and variations thereof such as "comprising", "containing" and "having" are not intended to exclude other additives, components, integers or steps.

[0034] Further aspects of the invention and further implementations of the aspects described in the preceding paragraphs will become apparent from the following description, which is given by way of example. DETAILED DESCRIPTION

[0035] In describing and claiming the present invention, the following terms are used according to the following definitions. It should also be understood that the terms used herein are only used to describe specific embodiments of the present invention and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0036] As used herein, the term "about" may mean within 1 or more standard deviations according to the practice in the art. Alternatively, "about" may mean a range of up to 20%. When specific values ​​are provided in the specification and claims, the meaning of "about" should be considered to be within an acceptable error range for that specific value.

[0037] In the context of the present invention, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, horses, cattle, dogs, etc. The subject population can be a population of a specific species or subspecies, such as humans suffering from a specific disease (such as AD, early-onset AD, late-onset AD, or AD with a deletion in the Uppsala APP form).

[0038] In the context of the present invention, the words "include", "including" and the like should be interpreted in an inclusive rather than exclusive sense, that is, the meaning of "including but not limited to".

[0039] The terms "preferred" and "preferable" refer to embodiments of the present invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the description of one or more preferred embodiments does not mean that other embodiments are useless, and is not intended to exclude other embodiments from the scope of the present invention.

[0040] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about".

[0041] The recitation of numerical ranges using endpoints includes all numbers within that range (eg, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0042] As used herein, the term "acetylglucosamine" refers to glucosamine in which one or more -OH groups are replaced by -OAc (i.e., acetoxy, also shown as CH3-C(=O)-O-). Optionally, acetylglucosamine has one acetylation at one of R1, R2, R3, or R4. Optionally, acetylglucosamine has two acetylations at one of R1 and R2, R1 and R3, R1 and R4, R2 and R3, R2 and R4, or R3 and R4. Optionally, acetylglucosamine has three acetylations at one of R1, R2 and R3, R2, R3 and R4, R1, R3 and R4, or R1, R2 and R4.

[0043] As used herein, the term "tetraacetyl glucosamine" (TAG) refers to glucosamine in which all -OH groups have been replaced with -OAc.

[0044] The scope of the present invention includes salts and isomers of acetylglucosamine. The term "salt" as used herein means acidic salts and / or basic salts formed with inorganic acids and bases and / or organic acids and bases. Quaternary ammonium salts, such as alkylammonium salts, are also included herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred. Salts of compounds can be formed, for example, by reacting the compound with an amount of acid or base (e.g., an equivalent) in a medium (e.g., a medium in which the salt is precipitated) or in an aqueous medium, followed by lyophilization.

[0045] Exemplary salts include ammonium salts, alkali metal salts (such as sodium salts, lithium salts and potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts), salts formed with organic bases (e.g., organic amines) (such as benzathine penicillin, dicyclohexylamine, hydrabamine, N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butylamine) and salts formed with amino acids (such as arginine, lysine, etc.).

[0046] Solvates of the compounds of the present invention are also contemplated herein. The solvates of acetylglucosamine are preferably hydrates or other pharmaceutically acceptable solvates.

[0047] All stereoisomers of acetylglucosamine, such as those that may exist due to asymmetric carbons on the R substituents of the compound, including enantiomeric and diastereomeric forms, are within the scope of the present invention. Individual stereoisomers of the compounds of the present invention may, for example, be substantially free of other isomers, or may be mixed, for example, as racemates or mixed with all other or other selected stereoisomers. The chiral centers of the present invention may have an S or R configuration.

[0048] As used herein, the term "treatment" or "therapy" includes reversing, reducing or suppressing the symptoms, clinical signs and potential pathology of the patient's condition, to improve or stabilize the patient's condition. As used herein, and as known in the art, "treatment" is a method of obtaining a beneficial or desired result (including clinical results). Beneficial or desired clinical results may include but are not limited to relieving or improving one or more symptoms or conditions, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease state, preventing disease spread, delaying or slowing disease progression, improving or alleviating the disease state and alleviating (whether partially or completely), whether it is detectable or undetectable. "Treatment" can also refer to, compared with the expected survival period without treatment, prolonging the survival period. Treatment is optionally treating cognitive impairment.

[0049] Treatment can also be treatment of a preclinical cognitive condition. For example, treatment is of a preclinical condition that is expected to develop into cognitive impairment. Optionally, the preclinical cognitive condition is mild cognitive impairment. Optionally, the subject with the preclinical condition also has biomarkers that are expected to develop cognitive impairment. Optionally, the preclinical cognitive condition is determined based on a screening test for cognitive impairment, suitable tests are described below, or is determined by functional neuroimaging, suitable imaging is described below.

[0050] As used herein, "prevent" or "preventing" is intended to mean at least reducing the likelihood of a risk (or susceptibility) of developing a disease or condition (i.e., causing a subject who may be exposed to or susceptible to the disease but has not yet experienced or displayed symptoms of the disease to not develop at least one clinical symptom of the disease). Biological and physiological parameters for identifying such subjects are provided herein and are also well known to physicians. For example, preventive or prophylactic measures may be taken for patients deemed to be at risk (e.g., patients determined to suffer from dementia or other cognitive or neurodegenerative diseases or other conditions described herein).

[0051] Alternatively, prevention of cognitive impairment or prevention of preclinical cognitive conditions occurs in an object identified as having a risk of cognitive impairment or preclinical cognitive conditions. Alternatively, the object at risk of cognitive impairment is an object with preclinical cognitive conditions. Alternatively, the object at risk of cognitive impairment or preclinical cognitive conditions has one or more of the following: (i) family history of cognitive impairment and / or genetic risk factors for cognitive impairment; (ii) biomarkers of cognitive impairment. Alternatively, the object at risk of cognitive impairment does not have symptoms of cognitive impairment. Genetic risk factors for cognitive impairment (such as AD) are known in the art. In addition, it is expected that genetic risk factors will be identified over time, and it is considered that objects with such genetic risk factors are treated / prevented. Similarly, cognitive biomarkers such as AD are known in the art. In addition, it is expected that genetic risk factors will be identified over time, and it is considered that objects with such biomarkers are treated / prevented.

[0052] The compounds of the present invention can be used in pharmaceutical compositions, for example, in combination with a pharmaceutically acceptable carrier for administration to a patient. Such compositions may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, and other substances known in the art. The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. Such additional factors and / or agents may be included in the pharmaceutical composition to produce a synergistic effect with the compounds of the present invention, or to minimize the side effects caused by the compounds of the present invention. The pharmaceutical composition is optionally in the form of a solid, preferably a tablet, capsule, or powder. The preparation of these dosage forms is known in the art.

[0053] The pharmaceutical composition of the present invention can be in the form of liposomes or micelles, wherein the compound of the present invention is combined with an amphipathic agent (e.g., lipid) in addition to other pharmaceutically acceptable carriers, and is present in an aqueous solution in an aggregated form as a micelle, an insoluble monolayer, a liquid crystal, or a lamellar layer. Lipids suitable for liposome preparations include, but are not limited to, monoglycerides, diglycerides, glucosinolates, lysolecithin, phospholipids, saponins, bile acids, etc. The preparation of such liposome preparations is within the technical level of the art, for example, as described in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference. These pharmaceutical compositions are optionally liquid.

[0054] The compositions can be administered in a variety of ways, including orally, nasally, buccally, sublingually, intravenously, transmucosally, parenterally, by inhalation, spray, transdermally, subcutaneously, intrathecally, topically, and rectally, and can be formulated according to methods known in the art.

[0055] The effective dose of acetylglucosamine in the mouse model is expected to be about 1000 to about 2000 mg / kg active compound / body weight, which can be administered as a single dose or in separate doses, for example 1 to 4 times a day. The human equivalent dose (HED) can be inferred using recognized methods (Nair & Jacob 2016, J Basic Clin Pharm; 7(2): 27–31; Regan-Shaw et al. 2007, FASEB J 22: 659-662).

[0056] As used herein, the term "cognitive disease" refers to a disease characterized by decreased or impaired cognitive function. Cognitive function covers a variety of psychological processes, including memory, attention, language, perception, problem solving and decision making. Cognitive disease can manifest in different ways and has various potential causes. Some common cognitive diseases include AD, vascular dementia, Parkinson's disease dementia, Lewy body dementia, frontotemporal dementia and mild cognitive impairment (MCI). These conditions can affect individuals of different ages, but are more common in the elderly. Symptoms of cognitive disease can include memory loss, language and communication difficulties, difficulty in reasoning and problem solving, impaired judgment, personality changes and difficulty in daily tasks.

[0057] As used herein, the term "Alzheimer's disease" (AD) is a progressive neurodegenerative disease characterized by cognitive decline, memory loss, and behavioral changes. It is characterized by the accumulation of abnormal protein structures in the brain, such as beta-amyloid plaques and tau tangles. Early-onset AD refers to a specific form of AD that occurs before the age of 65 years, accounting for approximately 5% to 10% of all AD cases. Late-onset AD is the most common form of AD, usually occurring after the age of 65 years. It accounts for approximately 90% to 95% of all diagnoses. The Uppsala APP deletion form of AD, also known as the "Swedish mutation," refers to an autosomal dominant deletion (Δ690–695) that causes AD. Affected individuals have early onset, with symptoms appearing in their early forties, and the disease progresses rapidly.

[0058] As used herein, a subject with a "family history of cognitive impairment" may include an individual with at least one mutation in at least one gene known to be associated with AD or to cause AD. AD is a heterogeneous disease, and multiple gene mutations are known to be associated with AD, and have autosomal dominant inheritance. For example, genes encoding amyloid precursor protein (APP), presenilin (PSEN1) and / or presenilin 2 (PSEN2) mutate. Many such variants have been identified, and regions in which mutations in these proteins may be the cause of AD or may also be benign are well known in the art (Xiao et al. 2021, Front Aging Neurosci., 13: 695808). However, those skilled in the art will appreciate that specific mutations that have not yet been characterized may have effects similar to those previously described, are likely to be characterized in the future, and may also be present in individuals with a family history of AD. It is well known that variations that cause AD include AD in the form of Uppsala APP deletion.

[0059] As used herein, a subject with a "genetic risk factor for AD" may include individuals with a protein variant associated with an increased risk of developing AD. For example, individuals carrying the ApoE4 allele of apolipoprotein E have a 10 to 15 times greater risk of developing AD than individuals carrying the most common ApoE3 allele.

[0060] As used herein, a subject with a "biomarker of cognitive impairment" may include an individual who has changed at least one of the following: scores on psychometric screening tests, biomarkers in blood and / or cerebrospinal fluid, and / or measurements obtained by brain imaging, which are associated with AD. Biomarkers associated with AD are well known in the art and are discussed in further detail in

[0062] to

[0066] . Biomarkers can be used alone or in combination. For example, an assay for measuring the plasma Aβ42 / 40 ratio can be used in combination with PET imaging (Brand et al. 2022, Alzheimers Res Ther., 14 (1): 195). However, it will be appreciated by those skilled in the art that further methods can be developed to identify individuals at risk of developing AD.

[0061] As used herein, the terms "reduced expression", "reduced level" and the like refer to a decrease or lower level of the activity or production of a particular gene, protein, molecule or biological component compared to a normal or reference state.

[0062] As used herein, the terms "increased expression," "increased levels," and the like refer to an increase or higher level of the activity or production of a particular gene, protein, molecule, or biological component compared to a normal or reference state.

[0063] As used herein, the term "seizure-related death" refers to situations in which an individual dies as a result of an epileptic seizure or its complications. An epileptic seizure is an abnormal and excessive electrical activity in the brain that can result in a variety of physical and behavioral manifestations.

[0064] As used herein, the term "spatial working memory" refers to the cognitive abilities and temporary storage systems that allow a subject to hold and manipulate spatial information in the mind for short periods of time. It involves the mental representation, manipulation, and updating of spatial relationships and locations. Spatial working memory is essential for a variety of cognitive processes and daily activities that require navigation, orientation, and spatial reasoning. It helps individuals remember and manipulate spatial information in their minds, such as the location of objects, the layout of a physical environment, or the path to a destination.

[0065] A subject's spatial working memory may be measured by one or more of the following techniques to determine whether the subject suffers from a cognitive impairment, a preclinical cognitive condition, or is healthy.

[0066] In some embodiments, treating cognitive impairment can refer to a reduction in the score on the scale or subscale of the Neuropsychiatric Inventory-Nursing Home Version (NPI-NH) or the Alzheimer's Disease Collaborative Study-Clinical Global Impression of Change (ADSC-CGIC). For example, improvement refers to a reduction in the total NPI-NH score of a patient from 50 points to 40 points. In some embodiments, improvement can optionally refer to one or more patients.

[0067] In some embodiments, treatment of cognitive impairment can refer to the improvement of psychological screening test score.Prevention of cognitive impairment can refer to maintaining or improving the health or preclinical cognitive status score of the object, and / or preventing progression to cognitive impairment score (particularly when such progression is expected). The screening test for cognitive impairment can include 3MS, CASI, MMSE, SASSI, STMS, CAST, GPCOG, 7MS, AMT, Mini-Cog, SIS, T&C, ACE-R or DemTect, self-management tools (such as p-AD8+IQCODE) and / or test combinations (such as DemTect and MoCA, or MES and MoCA). For example, improvement can be the original score of the word list part of the DemTect test increased from 6 to 9. In some embodiments, improvement can alternatively refer to the result of one or more patients, or one or more tests, or colony. These tests can also be used to identify whether the object is healthy, suffers from preclinical disorder, or suffers from cognitive impairment before using acetylglucosamine.

[0068] In some embodiments, treating cognitive impairment can refer to improving cerebral blood flow and / or cerebral metabolism, and / or stopping the progression of clinical cortical atrophy detected by functional neuroimaging or reversing clinical cortical atrophy detected by functional neuroimaging. Preventing cognitive impairment can refer to improving healthy or preclinical levels of cerebral blood flow and / or cerebral metabolism, and / or stopping or reversing healthy or preclinical levels of cortical atrophy, or preventing cortical atrophy from progressing to levels of cognitive impairment (particularly when such progression is expected). Such imaging includes positron emission tomography (PET), fluorodeoxyglucose (FDG)-PET, 99m Tc-hexamethylpropyleneamine oxime (HMPAO), single photon emission computed tomography (SPECT) and / or magnetic resonance imaging (MRI) or a combination thereof, such as early frame Florabetaben PET / MRI. For example, the improvement can be an improvement in cerebral blood flow in a specific area of ​​the brain visualized using an imaging method. In some embodiments, the improvement can optionally refer to an improvement in one or more patients, and / or an improvement in one or more imaging methods.

[0069] In some embodiments, treating cognitive impairment can refer to stopping the progression of or reversing clinical changes in the level of a biomarker in cerebrospinal fluid (CSF). Preventing cognitive impairment can refer to improving healthy or preclinical levels of a biomarker, and / or stopping the progression of or reversing changes in preclinical levels or biomarkers (or biomarkers in patients in a preclinical state in screening or other testing), or preventing progression to clinical levels or biomarkers (or preventing progression to clinical results in screening or other testing in subjects with relevant biomarkers). Aβ42, tau, tau-p181, tau-p231, tau / Aβ 42 Ratio and synaptic protein NPTX2 are examples of CSF biomarkers for MCI, preclinical AD and AD. Reversal of changes in biomarker levels in CSF may lead to increased NPTX2 levels, thereby prolonging the onset of MCI symptoms. In some embodiments, improvement can optionally refer to one or more patients, or one or more biomarkers.

[0070] In some embodiments, treating cognitive impairment may refer to stopping the progress of biomarker levels in the blood or reversing its clinical changes. Preventing cognitive impairment may refer to improving the healthy level or preclinical level of a biomarker, and / or stopping the progress of preclinical levels or biomarkers (or biomarkers of patients in a preclinical state in screening or other tests) or reversing its changes, or preventing progression to clinical levels or biomarkers (or preventing progression to clinical results in screening or other tests in objects with relevant biomarkers). T-tau, tau-p181, NfL, AβPPr, Aβ42, Aβ42 / Aβ40 ratios and tau-p217 are examples of blood biomarkers for MCI, AD and AD progression. The reversal of changes in biomarker levels in the blood may cause NfL levels to decrease, thereby extending the onset time of MCI symptoms. In some embodiments, improvement may alternatively refer to one or more patients, or one or more biomarkers.

[0071] As used herein, treatment of clinical conditions may refer to a clinically relevant (desired) effect that is greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 300%, 400% or 500% compared to baseline after a specified time period. In some embodiments, treatment refers to an improvement in useful effects in a single patient compared to baseline (i.e., before administration). In other embodiments, treatment refers to a greater percentage of patients showing useful effects after a specified time period compared to placebo or lack of treatment. In various embodiments, the percentage of patients showing a useful effect is increased by greater than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 300%, 400%, or 500% compared to placebo or no treatment. In some embodiments, the specified time period is about two weeks, four weeks, or six weeks. In one embodiment, the specified time period is six weeks.

[0072] As used herein, treatment of a clinical situation may refer to a clinically relevant (unexpected) effect that is less than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 300%, 400% or 500% after a specified time period compared to baseline or compared to placebo or no treatment. In some embodiments, the specified time period is about two weeks, four weeks or six weeks. In one embodiment, the specified time period is six weeks.

[0073] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or apparent in the text. All these different combinations constitute various alternative aspects of the present invention. For the purpose of interpreting this specification, terms used in the singular also include the plural, and vice versa.

[0074] Now will be referred to in detail certain embodiments of the present invention. Although the present invention will be described in conjunction with the embodiments, it should be understood that its purpose is not to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the present invention defined by the claims.

[0075] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. The present invention is in no way limited to the methods and materials described. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the present invention.

[0076] All publications mentioned herein are incorporated by reference in their entirety.

[0077] Although the invention has been described with reference to certain embodiments described in detail herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be apparent to those skilled in the art, and the invention is intended to cover all such modifications and equivalents.

[0078] The inventors predict that in the brains of mice fed with acetylglucosamine, the expression of exostogen glycosyltransferase 1 (EXT1) and UDP N-acetylglucosamine will be downregulated. Both EXT1 and UDP N-acetylglucosamine are necessary for the synthesis of heparan sulfate, which is converted into a highly sulfated form in the AD brain (Wang et al. 2022, Anal Chem. 94 (6): 2950-2957; Perez-Lopez et al. 2021, J Neuropathol Exp Neurol. 80 (5): 446-456; Roberts 2017 et al. J Alzheimers Dis Rep. 1 (1): 115-124). During the biosynthesis of heparan sulfate, the polymer chain undergoes a series of enzymatic modifications, including a variety of O-sulfation of sugar chains. Highly sulfated heparan sulfate can include heparan sulfate with a higher degree of sulfation than the average level. Optionally, the highly sulfated heparan sulfate has increased levels of 3-O or 6-O sulfation in the heparan sulfate (Wang et al. 2023, Sci Adv., 9(21):eadf6232; Rauch et al. 2018, Sci Rep., 8(1):6382), and / or an increased number of sulfations in specific subdomains of the heparan sulfate such that they contain continuous sulfated disaccharide clusters and / or trisulfated disaccharide clusters (e.g., -iduronic acid (2S)-glucosamine (NS,6S)-) structures. The level of highly sulfated heparan sulfate in biopsy tissue can be measured by a variety of methods including histochemical techniques (Snow et al. 2021, Front Aging Neurosci., 13: 710683) and by determining heparan sulfate disaccharide composition and ratio (Karamanos et al. 1997, J Chromatogr A., ​​765 (2): 169-79). Antibodies that recognize highly sulfated heparan sulfate are also known in the art and include antibodies such as RB4CD12 antibodies (Hosono-Fukao et al. 2012, Am J Pathol., 180 (5): 2056-67. Methods for measuring EXT1 expression in biopsy tissue are also known in the art and can be measured using commercial ELISA kits and / or by quantitative PCR. Methods for measuring UDP-N-acetylglucosamine are well known in the art, including enzyme-based assays (Sunden et al, 2023, Cell Rep Methods, 3 (7): 100518).

[0079] The present inventors found that in an AD mouse model fed a diet supplemented with TAG, plasma glutamate levels were decreased, the ratio of glutamine / glutamate in plasma was increased, and UDP N-acetylglucosamine in brain tissue was increased.

[0080] The inventors anticipate that feeding acetylglucosamine will reduce seizure-related mortality and memory deficits in a mouse model of Alzheimer's disease.

[0081] The inventors found that memory deficits were reduced in AD mouse models fed a diet supplemented with TAG.

[0082] In another embodiment, a kit or article of manufacture comprising one or more acetylglucosamines is provided for use in any of the above methods according to the present invention.

[0083] In other embodiments, a kit for the above-mentioned therapeutic or preventive application is provided, the kit comprising:

[0084] - a container containing the acetyl glucosamine pharmaceutical composition of the present invention;

[0085] - Label or package insert with instructions for use.

[0086] The kit or "article of manufacture" may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be made of a variety of materials, such as glass or plastic. The container contains a therapeutic composition effective for treating a cognitive disease. The label or package insert indicates that the therapeutic composition is used to treat a selected cognitive disease. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or preventive composition can be used to treat a cognitive disease described herein.

[0087] The invention is further described by the following non-limiting examples.

[0088] Example

[0089] Example 1A - Expression of exoskeletal glycosyltransferase 1 in the brain of mice fed tetraacetyl glucosamine (TAG)

[0090] Exostrin glycosyltransferase 1 (EXT1) is a transmembrane glycosyltransferase involved in the chain elongation step of heparan sulfate biosynthesis. It catalyzes the glycosidic addition of N-acetylglucosamine and glucuronic acid to HS chains (derived from UDP N-acetylglucosamine).

[0091] Wild-type mice will be fed a TAG-supplemented diet or a control diet and then sacrificed by nitrogen asphyxiation. The brain removed by craniotomy will be cut in half along the sagittal plane, and one half of each mouse will be frozen and stored at -80°C. The brain tissue will be thawed, weighed, and total RNA will be extracted using a QIAGEN extraction kit with DNAse treatment. RNA will be quantified by absorbance at 260nm, and cDNA will be prepared by reverse transcription of equal amounts (585ng) of purified DNA, followed by amplification with random hexamers. QPCR for EXT1 will be performed using primers and fluorescent probes (Fluorescein Amidite) from IDT DNA. QPCR for 2 housekeeping genes will be performed in the same reaction using probes containing different fluorophores: hexachlorofluorescein probe for ribosomal protein LP0 QPCR and Cyanine 5 probe for Beta 2 macroglobulin QPCR.

[0092] EXT1 levels will be compared by the ddct method (Livak & Schmittgen 2001, Methods 25(4):402-408.) ddcT values ​​will be compared by one-tailed T-test.

[0093] Using the B2M housekeeping standard, it was expected that EXT1 expression would be reduced in mice fed TAG.

[0094] Example 1B - TAG reduces plasma glutamate levels and increases the glutamine / glutamate ratio in plasma of AD mice

[0095] In the healthy brain, the neurotransmitter L-glutamate is actively taken up from synapses by astrocytes and then converted to glutamine by glutamine synthase (GS). Impaired glutamine / glutamate cycling produces excess glutamate, which is excitotoxic to neuronal cells. The excitotoxic calcium ion leak in AD mice can be attributed to glutamate binding to defective Gria2AMPA2 receptors. Multiple studies have shown that impaired glutamine / glutamate metabolism is associated with AD etiology, including reduced glutamate transporter and glutamine synthase activity. Meta-analysis of clinical AD data, in vitro modeling, and animal modeling suggests that circulating glutamine has a protective effect against AD.

[0096] For more than two decades, transgenic mice have been used to study the role of genes in AD. The most widely used one is the TG2576 APPswe mouse (G2576), which expresses the APP695 variant of human amyloid precursor protein (h-APP) under the hamster prion protein (prp) promoter (Hsiao et al. 1996, Science 274 (5284): 99-102). APP695 carries the double mutation K670N-M671L, which is cleaved into amyloidogenic amyloid protein β1-42 peptide (Aβ1-42) in vivo. Hemizygous TG2576 mice will develop pathology similar to Alzheimer's disease, including amyloid neuropathology and cognitive deficits that affect spatial learning and working memory (Hsiao et al. 1996, Science 274 (5284): 99-102).

[0097] TG2576 mice were bred and maintained in a dedicated, PC2-licensed animal house with a 12:12 h light-dark cycle. Mice were fed a standard mouse chow diet and drinking water ad libitum. The TG2576 strain was maintained on a B6 / SJL background. Mice were genotyped by PCR of genomic DNA from mouse skin samples. Housing and care of animals was in accordance with NSW DPI ARRP guidelines.

[0098] Powdered rat and mouse feed pellets (Specialty Feeds, Canningvale, WA) were manually blended with 0.84% ​​(w / w) TAG. Sterile deionized water was then blended with the mixture at 10% w / w. The feed paste was ground through a pellet mill, and the pellets were baked at 75°C for 2.5 hours and then stored dry at -20°C until ready for use.

[0099] TG2576 mice were fed a TAG-supplemented diet or a control diet for 6 weeks. After euthanasia, core blood was collected by ventricular venipuncture. Plasma was prepared by centrifugation of whole blood. Plasma was diluted 1:20 with water and then passed through a molecular weight filter to remove high molecular weight contaminants (>1 kDa). 20 μl of sample was analyzed by LC-MS using an HPLC column (Phenomenex, Luna Phenyl-Hexyl 5 μm, 250 mm x 4.6 mm id) with the following gradient at 1 mL / min: isocratic elution with 1 mM ammonium formate (pH 4.3) for 4 minutes, followed by a linear gradient of 45% acetonitrile in 2 minutes to 1 mM ammonium formate (pH 4.3), maintained for 2 minutes. The Thermo LTQXL MS was operated in positive ion mode, scanning m / z 147 (M + H) parent ion.

[0100] The retention time was 2.7 minutes and there was a subfragment m / z 130 (M + Glutamine was identified using 1 mL of 90% acetonitrile and 0.1% formic acid (H-NH3). Glutamic acid was detected using the same LC column with an isocratic elution of 1 mL of 90% acetonitrile and 0.1% formic acid for 1 min, followed by a linear gradient to 5% MeCN, 5% MeOH, 0.1% formic acid over 4 min, maintained for 1.5 min. The Thermo LTQXL MS was operated in positive ion mode, scanning m / z 148 (M + The parent ion of H) was detected by the presence of a subfragment at m / z 130 (M + Glutamine was identified by 1:1 ratio of 1:1 (H-H2O). Glutamine / glutamate concentrations were calculated by regression analysis of sample peak integrals against a standard curve prepared using commercially available standards. Table 1 compares the control diet and the TAG-supplemented diet after 43 weeks of supplementation (age 51.8 ± 1.5 weeks at the time of plasma sampling) by a one-tailed t-test.

[0101] Table 1: Changes in plasma glutamate concentration, glutamine concentration, and glutamine to glutamate ratio after TAG supplementation

[0102]

[0103] Table 1 shows the effect of a 6-week diet supplemented with TAG on the concentration of glutamate, glutamine, and the ratio of glutamine to glutamate in the plasma of TG2576 mice. Glutamate and glutamine levels are expressed as RU / ml values ​​(recovery integral units / ml). The glutamate content of mice supplemented with TAG in the diet was significantly lower than that of control mice (p=0.046). The glutamine / glutamate ratio of mice supplemented with TAG in the diet was significantly higher than that of control mice (p=0.043).

[0104] Plasma glutamine trended toward increase in TG2576 mice on a TAG diet for 43 weeks, with a corresponding decrease in glutamate. The trend toward higher plasma glutamine levels, significantly lower glutamate levels, and a significantly higher glutamine / glutamate ratio suggest that dietary supplementation with TAG could be used to protect against AD-associated excitotoxicity.

[0105] Example 2A - Effect of TAG on glucose metabolism in AD

[0106] TG2576 mice will be bred and maintained in a dedicated, PC2-licensed animal room with a 12:12 h light-dark cycle. Mice will have ad libitum access to standard mouse chow and drinking water. The TG2576 strain is maintained on a B6 / SJL background. Mice will be genotyped by PCR testing of genomic DNA from mouse skin samples. Housing and care of animals will comply with NSW DPIARRP guidelines.

[0107] Powdered rat and mouse feed pellets (Specialty Feeds, Canningvale, WA) were manually blended with 0.84% ​​(w / w) TAG. Sterile deionized water was then blended with the mixture at 10% w / w. The feed paste was ground through a pellet mill, and the pellets were baked at 75°C for 2.5 hours and then stored dry at -20°C until ready for use.

[0108] Unperfused brains were obtained from euthanized animals by craniotomy. The brains were cut into two halves in the sagittal direction, one half was fixed with 10% paraformaldehyde / PBS, and the other half was frozen and stored at -80°C.

[0109] The fresh weight of the brain was measured, and the tissue was homogenized in 10 volumes of extraction buffer (methanol: acetonitrile: water = 5: 3: 2) using an Omni TH02 homogenizer at medium settings for 15 seconds. Before homogenization, the internal standard of 13C6 glucose-6-phosphate (620ng in 50μl buffer) was added to each extract. The crude extract was dispensed into 2x1.5ml labeled Eppendorf tubes and then centrifuged (4°C) for 15 minutes at 16000g. The supernatant was processed at 4000rpm for 30 minutes using a 1kDa centrifugal mwt cutoff filter (PALL Corp, MCP001C41). The low mwt fraction was transferred to a 2x1.5ml labeled microcentrifuge tube and then evaporated to dryness under reduced pressure using a centrifugal evaporator (Savant SC110, connected to a Dynavac FDA steam trap and Edwards M5 vacuum pump). The sample was stored at -20°C. One tube will be processed by solid phase extraction cleanup. SPE has a low analyte recovery, so most analytes will be analyzed as crude extracts. The SPE processed samples will be used to measure UDP sugars because they give high signals in MSMS and can therefore be detected in the SPE processed samples. The extracts for UDPN-acetylglucosamine and UDPN-acetylgalactosamine LC-MS / MS analysis will be purified by SPE as follows. The extracts will be re-dissolved at 37°C for 30 minutes by adding 100 μl acetonitrile / H2O, 1:1 (v / v). 1 ml of 2% formic acid will then be added to each sample. The SPE column (3cc OASIS WAX, Waters) will be activated by loading 2 ml of methanol and then washed with 2 ml of 2% formic acid. The extract will be loaded and then washed with 2 ml of 2% formic acid and then with 2 ml of methanol. The acidic fraction will be eluted with 2 ml of 5% ammonia in methanol. The eluate will be dried under reduced pressure and stored at -20°C. Additional LC-MS / MS analysis will be performed on extracts not subjected to SPE.

[0110] For LC-MS, glucose-6-phosphate, fructose-6-phosphate, 6-phosphogluconic acid, ribulose-5-phosphate, xylulose-5-phosphate, ribose-5-phosphate, erythrose-4-phosphate, sedoheptulose-7-phosphate, glucosamine-6-phosphate, N-acetylglucosamine-6-phosphate, UDP N-acetylglucosamine, and UDP N-acetylgalactosamine will be purchased from Sigma Aldrich. 13C6 glucose-6-phosphate (>99%) will be purchased from Cambridge Isotope Laboratories.

[0111] The levels of glucosamine-6-phosphate and fructose-6-phosphate can also be measured using commercially available fluorescence assay kits (Abcam).

[0112] The degassed and filtered mobile phase will be delivered isocratically or in gradient at 1 ml / min through a Shimadzu SCL-10A system controller and LC-10AD liquid chromatograph. The tube containing 50% of the total hemibrain extract was reconstituted in 50% acetonitrile water at 37°C for 15 minutes. 40 μl of the extract in a total volume of 100 μl was added to 90 μl of mobile phase A and then injected into a 100 μl injection loop.

[0113] LC-MS of glucosamine-6-phosphate, erythrose-4-phosphate and fructose-1,6-bisphosphate will be performed on a mixed mode hydrophilic HPLC column (150 mm x 4.6 mm id) with a guard column (Newcrom B, provided by SIELC) ​​using the following gradient: 20% MeCN / 0.3% formic acid (A) for 10 minutes, then linearly ramped to 100% solvent B (10% MeCN 20 mM ammonium formate pH 3.3) in 1 minute. The mobile phase will be held at 100% B for 4 minutes, then returned to 100% A in 1 minute. Between samples, the column will be re-equilibrated for 10 minutes. LCMS of UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine will be performed on a mixed mode HPLC column (150 mm x 4.6 mm id) with a guard column (Primesep N, supplied by SIELC) ​​using isocratic 80% acetonitrile and 10 mM ammonium formate, pH 4.3. LC-MS of glucose-6-phosphate, fructose-6-phosphate, 6-phosphogluconic acid, pentose-5-phosphate and sedoheptulose-7-phosphate will be performed on a Newcrom B column using 20% ​​acetonitrile and 0.3% formic acid.

[0114] The HPLC column eluent will be split at a 5:1 ratio and injected via ESI into a Thermo-Finnigan LCQ Deca mass spectrometer operating in ESI negative ion mode with MS / MS detection. The MS / MS mode will be used to accumulate parent ions of specific mass / charge in the ion trap to react with the collision gas (He) to generate fragment ions used to identify the analytes. For all analytes, except for glucosamine-6-phosphate and fructose-1,6-bisphosphate, the fragment ion used for identification is m / z 97 (phosphate), which is the most abundant ion. Parent ions will be used for Gln 6P and m / z 385 for F16bp.

[0115] Xcalibur (version 2.0SUR1) will be used to analyze the chromatogram and integrate the analyte peak area. Peak integration will be converted to nanomolar amount by regression analysis relative to high purity standards run under the same conditions. Nanomolar amount will be standardized by correction of tissue fresh weight and internal standard 13C G6P. Group mean values ​​and standard errors (SE) and single-tailed T tests will be calculated using MS Excel software.

[0116] For RT-PCR analysis, brains will be thawed to 2 to 8 °C and then homogenized in QIAZOL RNA extraction solution (QIAGEN) using an Omni TH02 homogenizer. Total RNA will be extracted using an RNeasy solid phase cartridge (QIAGEN) with on-column DNAse digestion. RNA will be quantified by absorbance at 260 nm, and equal amounts of total RNA from all samples will be converted to cDNA using a commercial kit (RT2 First Strand kit, QIAGEN) and a Rotorgene Q thermal cycler (QIAGEN). Vendor-validated PCR assays, including primers and fluorescent probes, will be purchased from IDTDNA. ddCT RT-PCR will be performed using 2 housekeeping genes. Reactions will be loaded using a QIAgility (QIAGEN) robot and thermal cycling will be performed using Rotorgen Q, setting up triple PCR runs. Changes in expression levels will be analyzed by ddCT analysis, and a one-tailed t-test will be used to compare the means of TAG and control diets.

[0117] For blood chemistry, whole core blood will be drawn from the ventricle using an 18-gauge needle and heparin will be added to prevent clotting. Plasma will then be prepared by centrifugation to remove red and white blood cells. Whole plasma will be stored frozen at -20°C. 50 μl of whole plasma will be loaded into a blood chemistry analyzer (Spotchem SP 443, Arkray) equipped with a diagnostic test strip (Arkray).

[0118] The peak integrated area will be converted to x10 6, and then the peak integral is converted to nanomolar amount using regression analysis of the standard curve. The nanomolar signal will be corrected according to the injection amount. A conversion factor of 1 / 0.154=6.49 will be applied to obtain the total nanomolar analyte per hemibrain. The amount of the whole extract will be corrected according to the reconstituted internal standard (IS) (620ng of 13C6G6P is added to each hemibrain). The amount of 13C G6P nanomoles / hemibrain will be calculated by regression of the MSM signal of 13C6 G6P (fragment m / z 97 in the parent m / z 265). The ratio of added and reconstituted 13C6 G6P will be applied to the value of nanomoles per hemibrain. These values ​​will be standardized by dividing the nanomoles / mg value of each analyte by the measured fresh weight of the hemibrain according to the change in the amount of tissue extracted. A one-tailed Student's t-test will be used to compare the mean values ​​(n=5-6) grouped by genotype (WT vs. TG2576) and experimental diet (control vs. TAG).

[0119] It is expected that the content of glucosamine-6-phosphate and glucose-6-phosphate in mice fed with TAG will increase. It is expected that the level of fructose-6-phosphate in mice fed with TAG will also increase, which can be explained by the increased availability of glucose-6-phosphate. The reaction is catalyzed by glucose phosphotisomerase (GPI). GPI is also a "part-time" protein that, when secreted, can act as an extracellular cytokine and autocrine motility factor called neuroleucin (Haga 2000). GPI has a neuroprotective effect (Knight et al. 2014, Cell Metab. 20 (1): 145-57), and its expression is downregulated in the brain tissue of AD patients (Li et al. 2021, Heregitas 158 (1): 25), and increased in the cerebrospinal fluid of AD patients (De Kort et al 2021, Alzheimers Res Ther. 13 (1): 160). There is a receptor for GPI, called gp78 / AMFR E3 ubiquitin ligase, which is part of the endoplasmic reticulum degradation system that targets misfolded proteins and is associated with certain neurodegenerative diseases (Huntington's disease, not AD). If GPI is deficient in AD due to its moonlighting neuroprotective role, the formation of fructose-6-phosphate for glycolysis would be reduced. Increasing the production of fructose-6-phosphate by administering TAG could compensate for the lack of GPI.

[0120] Example 2B - Effect of dietary TAG on UDP N-acetylglucosamine in brain tissue of AD mice

[0121] Both EXT1 and uridine diphosphate N-acetylglucosamine (UDP N-acetylglucosamine) are required for the synthesis of heparan sulfate, which is then converted to a highly sulfated form in AD brain. EXT1 is a transmembrane glycosyltransferase involved in the chain elongation step of heparan sulfate biosynthesis. It catalyzes the glycosidic addition of N-acetylglucosamine and glucuronic acid to heparan sulfate chains (from UDP N-acetylglucosamine).

[0122] UDP N-acetylglucosamine is a biosynthetic precursor in the chain elongation reaction of heparan sulfate biosynthesis, which is carried out by the heterodimeric enzymes EXT1 / EXT2 (EC: 2.4.1.224, EC: 2.4.1.224):

[0123] UDP N-acetylglucosamine + heparan sulfate <=> UDP + (N-acetyl-D-glucosamine) - heparan sulfate

[0124] The next step in chain elongation is the addition of glucuronic acid via EXT1 / EXT2:

[0125] UDP-glucuronic acid + heparan sulfate <=> UDP + (glucuronic acid) - heparan sulfate

[0126] UDP N-acetylglucosamine is also an allosteric inhibitor of glutamine fructose-6-phosphate aminotransferase or GFAT (EC: 2.6.1.16), the enzyme that catalyzes the hexosamine pathway reaction:

[0127] Fructose-6-phosphate + glutamine <=> glucosamine-6-phosphate + glutamic acid

[0128] Given the effects of TAG on plasma glutamate / glutamine levels in TG2576 AD mice (Example 1) and the role of UDPN-acetylglucosamine in heparan sulfate chain modification in the brain (as described above), the inventors sought to determine whether UDP N-acetylglucosamine levels would be affected by dietary supplementation with TAG.

[0129] To study the effect of TAG on UDP N-acetylglucosamine levels, adult male and female TG2576 mice (>8 weeks old) were fed standard chow (n=5) as a control diet or batch-matched chow supplemented with 0.45% (w / w) TAG (n=5). Six weeks later, the brain was removed and cut in half along the sagittal plane. The hemibrains were stored frozen (-80°C), then thawed, weighed and homogenized in 10 volumes of MeOH / MeCN / H2O (50% / 30% / 20%, v / v / v). The homogenate was centrifuged at 5000rpm for 30 minutes. The supernatant was passed through a 1 kDa MW centrifugal filter, then evaporated and resuspended in HPLC mobile phase. UDP N-acetylglucosamine was run on a mixed mode hydrophilic HPLC column (150 mm x 4.6 mm id, Primesep N, provided by SIELC) ​​using a guard column under the following linear gradient: from 80% acetonitrile and 10 mM ammonium formate pH 4.3 to 10% MeCN and 10 mM ammonium formate in 9 minutes, and then returned to the starting conditions in 1 minute. UDP N-acetylglucosamine in brain extracts was quantified relative to commercial standard UDP N-acetylglucosamine by mass spectrometry (MS-MS) in negative ion mode, scanning m / z 606 and identifying the main collision fragments m / z 385 and 403. UDP levels were corrected for brain mass. A one-tailed t-test was used to calculate the effect of diet. Table 2 compares the levels of UDP N-acetylglucosamine in the brain of TG2576 mice fed a TAG-supplemented diet or a control diet after 6 weeks (age 21.2 ± 1.5 weeks at the time of brain sampling).

[0130] Table 2: UDP N-acetylglucosamine levels in the brain of TG2576 mice after 6 weeks of TAG-supplemented diet.

[0131]

[0132] After 6 weeks of a TAG-supplemented diet, UDP N-acetylglucosamine levels increased by 40% in the brain of TG2576 mice. UDP N-acetylglucosamine is an inhibitor of the enzyme GFAT, so the observed increase may explain the decrease in glutamate plasma and the increase in the glutamine / glutamate ratio, both of which are dependent on GFAT activity. UDP N-acetylglucosamine is also a required substrate for heparan sulfate chain elongation by EXT1 / EXT2. Recent studies on EXT1 / EXT2 activity and its effects on chain modifications suggest that increased UDP N-acetylglucosamine in the brain may be associated with altered sulfation patterns of brain heparan sulfate.

[0133] Heparan sulfate is known to co-deposit with Aβ plaques, bind to and accelerate Aβ aggregation, and mediate in vivo cytotoxicity, Aβ clearance, and neuroinflammation. The inventors set out to test whether a diet supplemented with TAG and its potential effects on heparan sulfate processing in the brain could reduce AD-associated memory deficits.

[0134] Example 3A - Effects of dietary TAGs on AD memory and lifespan

[0135] The T-maze has been used to simply and sensitively detect spatial working memory deficits in TG2576 mice (Lalonde et al. 2003, Brain Res 977(1):38-45; Stewart et al. 2011, J Alzheimers Dis 26(1):105-26). When a mouse is placed at the bottom of a T-maze, it moves along a path to the junction of the left and right arms at the far end of the maze, at which point it can choose to move along either the left or right arm. If the same mouse is then immediately placed back into the T-maze, it will instinctively tend to choose the arm opposite to the one it previously chose. This tendency is called spontaneous alternation and is interpreted as an exploratory behavior that mice use to find food, water, mates, or shelter (Dember and Fowler 1958, Psychological Bulletin 55(6), 412–428, Montgomery 1952, Journal of Experimental Psychology 42(2), 82–93). The frequency of spontaneous alternation can be increased by using food-deprived subjects and food rewards (Deacon & Rawlins 2006, Nat Protoc 1 (1): 7-12). During spontaneous alternation, mice use spatial working memory (Kraeuter et al. 2019, Methods Mol Biol 1916: 105-111) to distinguish between unexplored paths and previously explored paths. In these studies, the inventors used the T-maze to measure the efficacy of the dietary supplement TAG as a potential treatment for AD.

[0136] As in the previous examples, TG2576 mice were bred and maintained in a dedicated physical housing facility with a 12:12 h light-dark cycle and were fed ad libitum either standard mouse chow or TAG-supplemented chow. The food reward was 20 mg sucrose pellets (Fisher and Son, Somerville, NJ).

[0137] The T-maze was made of MDF board, 200 mm high, and consisted of a left arm, a right arm (350 x 100 mm), and a starting path (300 x 100 mm). Removable partitions used as barriers were located 220 mm (starting point) or 260 mm (left and right sides) from the end of each arm. A 70 mm central vertical board was placed at the junction of the left and right arms to improve the subject's spatial recognition ability. The interior was painted black.

[0138] Male and female mice were fed experimental diets for different time periods: standard mouse chow (control) or batch-matched chow supplemented with TAG. The average age of the mice in the study was at least 5 months. At this age, spatial working memory deficits in TG2576 mice become apparent (Westerman et al. 2002, J Neurosci 22(5):1858-67; Arendash et al. 2004, NeuroReport 15:1751-1754).

[0139] The treatment groups are TG2576 mice fed a control diet, WT mice fed a control diet, TG2576 mice fed a TAG diet, and WT mice fed a TAG diet. Each treatment group will contain 10 mice at the start of the experimental diet. In each study, mice will be assigned to each treatment group to make age and gender consistent. Mice will start the experimental diet when the average age is 11 weeks (range = 4-35) and will remain free to eat before the T maze test.

[0140] Prior to the T-maze test, mice will be acclimated to the maze for 4 days, allowed to explore the maze for 5 minutes each day, and reward pellets will be placed throughout the maze. The day before the T-maze test, mice will be provided with a short-term food ration of 2.5 g / day / mouse to improve T-maze performance (Corcoran et al. 2002, Learn Mem 9(5):243–252). During the T-maze test, mice will continue to eat the treatment diet. Weekly testing will include 4 consecutive days of T-maze testing, followed by 3 days of rest, no testing and ad libitum food. T-maze testing will continue for 4 weeks (16 test days).

[0141] On the testing day, each mouse will go through 5 consecutive T-maze trials. Each trial consists of a blind run, in which the entrance to one arm (L / R) of the T-maze will be blocked by an obstacle, and a reward pellet will be placed in the unblocked arm. The blind run will be immediately followed by an open run without obstacles, in which a food reward will be placed in the arm opposite to the position of the blind run. In this way, mice will be rewarded for alternating. In each trial, the position of the pellet in the right or left arm will be pseudo-randomized.

[0142] Each time the T-maze is run, a mouse will be placed behind a partition at the base of the maze. The partition will then be removed to allow entry into the maze. If the subject finds the reward during the start of the trial, the trial score will be scored as a success (score = 1). If the mouse returns to the correct arm containing the reward, it will be tolerated to move up to 100 mm to the wrong arm. The mouse will be allowed to walk in the maze for 1 minute. If the mouse moves slowly, it will be gently poked from behind to induce it to move. The maze between cages will be cleaned and dried using isopropyl alcohol (70% v / v) applied with a lint-free wipe.

[0143] For each T-maze testing day, the alternation rate for each mouse was calculated by converting the number of correct L / R choices (raw data, range = 0-5) to the percentage of correct alternations (Lalonde et al. 2003, Brain Res 977(1):38-45), as shown in Equation 1:

[0144]

[0145] For each treatment group, the mean alternation rate (MAR) will be calculated during the early, middle, and late phases of the T-maze test, corresponding to days 1-5, days 6-10, and days 11-16, respectively. T-tests will be used to compare the mean alternation rates between treatment groups.

[0146] During the course of the experiment, TG2576 hemizygotes are expected to die prematurely. To investigate the effects of dietary TAG, mortality data for TG2576 mice fed TAG or control diet mice will be pooled (4 cohorts, n=41) and plotted as Kaplan-Meier plots. TG2576 APP males will also be bred to C57B / 6 females, as this pairing has been reported to result in higher mortality in offspring (Carlson 1997).

[0147] It is expected that TG2576 mice will obtain lower MAR than WT, which is consistent with previous studies (Hsiao et al. 1996, Science 274(5284):99-102; Chapman et al. 1999, Nat Neurosci 2(3):271-276; Corcoran et al. 2002, Learn Mem 9(5):243–252; Lalonde et al. 2003, Brain Res 977(1):38-45). The increase in MAR values ​​over time suggests that both hemizygous and wild-type mice tend to achieve reward-induced alternation in the T maze.

[0148] It is expected that supplementation of TAG in the diet of TG2576 mice will increase MAR values ​​and survival.

[0149] Example 3B - Effects of dietary TAG on spatial working memory in AD

[0150] The T-maze consisted of a left arm, a right arm (350x100mm), and a starting path (300x100mm). A removable partition used as a barrier was located 220mm (starting point) or 260mm (left and right sides) from the end of each arm. A 70mm central vertical board was placed at the junction of the left and right arms to improve the subject's spatial recognition ability.

[0151] Adult male and female mice (mean age 14.3 ± 0.84 weeks) were fed an experimental diet: standard mouse chow (control) or batch-matched food supplemented with TAG. The treatment groups reported here are TG2576 mice fed a control diet, wild-type (WT) sibling mice fed a control diet, and TG2576 mice fed a 0.45% (w / w) TAG-supplemented diet. Diet was provided ad libitum before the T-maze test. During the T-maze test, mice were fed a treatment diet, and a short-term food ration of 2.5 g / day / mouse was provided for 4 consecutive days during the T-maze test (to improve T-maze performance), followed by 3 days of rest, no testing and ad libitum food.

[0152] The mean age of the mice in the study at the time of memory testing was 45 ± 0.35 weeks. At this age, the spatial working memory deficits of TG2576 mice became apparent. Prior to the T-maze test, the mice were acclimated to the maze for 4 days, and were allowed to explore the maze for 5 minutes each day with reward sucrose pellets placed throughout the maze. The T-maze test lasted for 4 weeks (16 testing days).

[0153] On the test day, each mouse was run through 5 consecutive T-maze trials. Each trial consisted of a blind run in which the entrance to one arm (L / R) of the T-maze was blocked by an obstacle, and a reward pellet was placed in the unblocked arm. The blind run was immediately followed by an open run without obstacles, in which a food reward was placed in the arm opposite to the position of the blind run. In this way, mice were rewarded for alternating behaviors. In each trial, the position of the pellet in the right or left arm was pseudo-randomized.

[0154] Each time the T maze is run, a mouse is placed behind a partition at the base of the maze. The partition is then removed to allow entry into the maze. If the subject finds the reward during the start of the trial, the trial score is scored as a success (score = 1). If the mouse returns to the correct arm containing the reward, it is tolerated to move up to 100 mm to the wrong arm. Allow the mouse to walk in the maze for 1 minute. If the mouse moves slowly, gently poke it from behind to induce it to move. Use a lint-free wipe to apply isopropyl alcohol (70% v / v) to clean and dry the maze between cages.

[0155] For each T-maze testing day, the alternation rate of each mouse was calculated by converting the number of correct L / R choices (raw data, range = 0-5) into the percentage of correct alternations (Lalonde et al. 2003, Brain Res 977(1):38-45), as shown in Equation 1:

[0156]

[0157] The effects of genotype and TAG diet on T-maze performance were compared using odds ratios. Results are expressed as odds ratios. Odds ratios (ORs) are a measure of the association between an exposure and an outcome. The OR represents the odds of an outcome occurring under a particular condition / exposure compared to the odds of that outcome occurring without that condition / exposure. An increase in odds is greater than 1; a decrease in odds is less than 1 (e.g., OR=2 means the odds have doubled; OR=0.5 means the odds have halved). If the 95% CI does not include 1, the odds are significantly different.

[0158] Using this model, the effects of genotype (WT vs. TG2576) and diet (control vs. TAG supplementation) on T-maze scores (0-5) were measured. The model was repeated in a subset of the timed data to obtain learning curves for mice that were unfamiliar with the maze.

[0159] The MAR values ​​of TG2576 mice were lower than those of their WT littermates, which is consistent with previous studies. After TAG was added to the diet of TG2576 mice, the MAR values ​​increased significantly after 16 days, as shown in Table 3.

[0160] Table 3: T-maze results after 16 days

[0161] Treatment (n) MAR ± SE Odds ratio (95% CI) p-value TG2576-control (7) 67.8±2.8 0.18vs WT(0.09,0.36) <0.001 WT-control diet (8) 79.4±2.8 n / a n / a TG2576-TAG Diet (8) 74.6±2.4 2.29 vs TG2576 control (1.16, 4.50) 0.017

[0162] Table 3 shows the effect of TAG diet on spatial working memory measured by using an alternating T-maze, with odds ratios, 95% confidence intervals (CIs), and statistical analysis. The results showed that in the TG2576 AD model, a diet supplemented with 0.45% (w / w) TAG had a significant (p=0.017) beneficial effect on memory deficits.

[0163] The Morris Water Maze (MWM) has been used to measure spatial memory deficits in TG2576 mice. Mice are placed in a bucket of water with a submerged platform inside. The time required for the mouse to swim to the platform is called the maze "escape time". The water maze has two testing phases: 1. a visible phase, in which the location of the platform is visible via a marker rod attached to the platform, and 2. a hidden phase, in which the marker rod is removed and the water is made opaque by the addition of white paint. Older TG2576 mice show increased latency to escape time compared to wild-type mice.

[0164] The maze consisted of a 715mm diameter barrel filled with 70mm of water. For the visible phase test, mice were placed in the water maze and the time they took to reach the platform was recorded with a stopwatch. This test was performed 8 times a day for 3 days, during which the platform was moved to various locations around the maze. The walls of the maze were engraved with 4 different shapes that served as spatial cues for learning. The hidden phase test lasted 7 days, 4 times a day. The location of the platform was fixed, but the mice were placed in a different location in the maze for each test.

[0165] The effects of genotype and TAG diet on Morris water maze performance were compared. Results are expressed as odds ratios. Odds ratios (ORs) are a measure of the association between an exposure and an outcome. The OR represents the odds of an outcome occurring under a particular condition / exposure compared to the odds of that outcome occurring without that condition / exposure. An increase in odds is greater than 1; a decrease in odds is less than 1 (e.g., OR=2 means the odds have doubled; OR=0.5 means the odds have halved). If the 95% CI does not include 1, the odds are significantly different.

[0166] Using this model, the effects of genotype (WT vs. TG2576) and diet (control vs. TAG supplementation) on water maze escape time were measured from days 2 to 7. The model was repeated in a subset of the timed data to obtain learning times for mice that were unfamiliar with the maze. As the subjects learned to navigate and find the hidden platform, their escape times decreased. Thus, in the water maze, escape time is inversely related to memory function.

[0167] As shown in Table 4, supplementation of the diet of TG2576 mice with TAG reduced the latency to escape after 7 days.

[0168] Table 4: Water maze results after 7 days

[0169]

[0170] Similar to the T-maze, the TAG-supplemented diet also significantly improved (p=0.037) spatial working memory as measured using the Morris water maze in the TG2576 mouse AD model. Taken together, these results demonstrate that dietary supplementation with TAG has significant benefits on memory function.

Claims

1. A method for treating cognitive impairment in a subject, comprising administering acetylglucosamine or a salt or hydrate thereof to the subject.

2. A method of treating a preclinical cognitive condition in a subject or preventing a cognitive disorder in a subject or preventing a preclinical cognitive condition in a subject, comprising administering acetylglucosamine to the subject.

3. The method of claim 2, wherein the method is a preventive method and the subject is identified as being at risk for developing a cognitive disorder or a preclinical cognitive condition.

4. The method according to claim 1, wherein the acetylglucosamine has the following structure: wherein at least one of R1, R2, R3 and R4 is an acetyl group, and the others are independently H or an acetyl group, or a pharmaceutically acceptable salt or hydrate thereof.

5. The method according to claim 3, wherein 1, 2, 3 or 4 of R1, R2, R3 and R4 of the acetylglucosamine are acetyl groups.

6. The method according to claim 3, wherein R1, R2, R3 and R4 are all acetyl groups.

7. The method of any one of claims 1 to 6, wherein a pharmaceutical composition comprising the acetylglucosamine, salt or hydrate and at least one pharmaceutically acceptable excipient is administered to the subject.

8. The method of any one of claims 1 to 7, wherein the acetylglucosamine, salt, hydrate or pharmaceutically acceptable composition is administered to the subject by a route selected from oral, nasal, buccal, sublingual, intravenous, transmucosal, parenteral, by inhalation, spray, transdermal, subcutaneous, intrathecal, topical and rectal.

9. The method of any one of claims 1 to 8, wherein the acetylglucosamine, salt, hydrate or composition is administered to the subject orally.

10. The method according to any one of claims 1 to 9, wherein the method reduces the expression of exosteoin glycosyltransferase 1 (EXT1) in the brain of the subject, or the method increases the expression of UDP N-acetylglucosamine in the brain of the subject, or the method reduces the level of highly sulfated heparan sulfate in the brain of the subject, or the method increases glucose-6-phosphate in the brain of the subject.

11. The method according to any one of claims 1 to 10, wherein the cognitive disorder is Alzheimer's disease (AD).

12. The method of claim 11, wherein the AD is selected from early-onset AD, late-onset AD, and Uppsala APP deletion form of AD.

13. The method of claim 11 or 12, wherein the method increases the level of glucosamine-6-phosphate in the brain of the subject.

14. The method of any one of claims 11 to 13, wherein the method increases the level of fructose-6-phosphate in the brain of the subject.

15. The method of any one of claims 11 to 14, wherein the method reduces seizure-related mortality in the subject.

16. The method of any one of claims 11 to 15, wherein the method reduces spatial working memory deficits in the subject.

17. Use of acetylglucosamine, a salt or a hydrate thereof in the manufacture of a medicament for treating cognitive impairment in a subject.

18. Use of acetylglucosamine, a salt or a hydrate thereof in the manufacture of a medicament for treating a preclinical cognitive condition in a subject or preventing a cognitive disorder in a subject or preventing a preclinical cognitive condition in a subject.

19. The use according to claim 18, wherein the use is for prevention and the subject is identified as being at risk of developing a cognitive disorder or a preclinical cognitive condition.

20. A kit comprising: - A container containing acetylglucosamine, its salt, hydrate, or pharmaceutical composition; -A label or package insert with instructions for use to treat cognitive impairment in a subject.

Images