WO2025076497A1 - Combination treatment of tau pathology - Google Patents

Abstract

A method for treating or preventing a tau pathology such as Alzheimer's disease in a subject in need thereof is described. The method includes administering a therapeutically effective amount of an amyloid-β receptor inhibitor in combination with a quercetin analog to the subject. Anti-tau pathology compositions including a therapeutically effective amount of an AβR inhibitor in combination with a quercetin analog are also described.

Description

COMBINATION TREATMENT OF TAU PATHOLOGY

GOVERNMENT FUNDING

[0001] This invention was made with government support under Grant No. RO1AG076699. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63/543,002, filed on October 6, 2023, which is incorporated herein by reference.

BACKGROUND

[0003] Alzheimer's disease (AD) stands as a prominent form of dementia in the United States, with approximately one million new cases diagnosed annually. Treatment for this condition remains highly elusive, with current therapeutic approaches limited to slowing progression or alleviating symptoms at best. Although numerous therapeutic agents have been investigated, most have failed to demonstrate efficacy in halting or reversing AD. It has been suggested that the failure of these treatments may be due, in part, to the presence of multiple etiological mechanisms that may operate in parallel. Importantly, recent research has been gradually unveiling the roles of tau proteins, which are found in an aggregated state within pathological tissues and cells, as well as the interplay and pathological associations they share with amyloid- P (Ap).

[0004] Aggregation of extracellular A peptides and intracellular neurofibrillary tangles of the microtubule-associated protein tau are the major pathological hallmarks of AD associated with degenerative brain changes. Although repeated failures of drug candidates targeting AD in clinical trials partially stem from an incomplete understanding of the molecular mechanisms underlying AD pathogenesis, recent research has gradually unveiled the critical role that tau proteins and their pathological aggregates have on cognitive function as well as their interplay and association with A . According to other recent reports, two potential tau aggregation mechanisms have been implicated in AD: an Ap-dependent pathway and an Ap-independent pathway.

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SUBSTITUTE SHEET (RULE 26) [0005] The A[3-dependent pathway has been previously suggested based on the observation that A[3 oligomers (A Os) can potentiate tau aggregation. ApOs demonstrate pronounced neurotoxic effects, concurrently with increased uptake mediated by Ap receptors (APR), which has been proposed as a pathway for cellular entry and tau dysfunction. One APR with nanomolar affinity to ApOs is human leukocyte immunoglobulin-like receptor B2 (LilrB2). The transport of ApO by this APR can be targeted to reduce tau uptake, cell-to-cell spreading, and aggregation. Still, inhibition of LiIrB2, while promising, will only decrease, but not prevent, the uptake of ApOs or other species that can potentiate tau aggregation. For this reason, the AP-independent pathway, where tau tangles form spontaneously or by the influence of other mechanisms, must also be considered.

SUMMARY OF THE INVENTION

[0006] Considering these two pathways, the inventors propose the investigation of a novel combination anti-tau therapy comprising a safe, repurposed drug (APR inhibitor) and a highly active flavonoid (Tau inhibitor). These components have been successfully identified through our in-silico to in-vivo motif-guided drug screening pipeline.

[0007] The inventors have addressed the challenge of AD treatment by focusing on the role of tau proteins and their pathological interactions with Ap. The outcome is the in-vivo demonstration of a novel, safe combination therapy for Alzheimer’s and tauopathy that targets both AP-dependent and -independent tau dysfunction and aggregation pathways. In addition to demonstrating the individual potentials of these compounds, this research will also validate the potential utility of a small molecule-based combination therapy targeting both tau and APR by providing a novel therapeutic approach that addresses various mechanisms contributing to AD onset and progression.

[0008] There are at least two important ways in which the combination therapy described herein is innovative. First, the CATT is based on the combination of safe plant-based products and repurposed old drugs. Drug repurposing is a desirable strategy for accelerating the discovery and approval of new treatments by making use of previously investigated or approved drugs with new targets. The method described herein combines previously identified drugs for repurposing with available, plant-based flavanols. While each of these compounds has been extensively studied in different areas, this is the first application of combination therapy as a highly selective intervention for targeting tau in Alzheimer’s disease (AD).

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SUBSTITUTE SHEET (RULE 26) Selection of the most effective agents has been guided by in-silico screening and in-vivo testing.

[0009] Second, combination therapy using the present method targets multiple pathways of tau aggregation. The amyloid hypothesis has been the mainstream explanation for AD pathogenesis for decades, but most of the prior clinical trials focused on amyloid burden reduction failed. Additionally, there are no current AD therapies that target tau. Recent research has implicated tau tangles as a probable neurotoxin culprit behind the neurodegeneration and cognitive decline observed in AD. Furthermore, preclinical evidence has also demonstrated that reductions in tau levels can reduce the impact of A0, supporting the notion that tau is central to the problem of AD.

BRIEF DESCRIPTION OF THE FIGURES

[0010] The present invention may be more readily understood by reference to the following drawings, wherein:

[0011] Figure 1 provides a schematic representation of the chemical structures of fluspirilene, myricetin, quercetagetin, and gossypetin.

[0012] Figure 2 provides a schematic representation of combination anti-tau therapy (CATT) using Amyloid- [3 receptor inhibitors and plant-based flavonols.

[0013] Figures 3A-3D provide graphs and images showing Ap oligomer potentiates tau aggregation in-vitro, a) fluorescent microscopy images of tau biosensor cells, b) Flow cytometry-based FRET quantification of tau aggregation in tau biosensor cells, c) fluorescent microscopy images of neuroblastoma, SH-SY5Y cells d) Flow cytometry-based FRET quantification e) A -promoted tau seeding diagram.

[0014] Figures 4A & 4B provide images showing a) LilrB2 crystal structure (PDB ID: 6BCS) and active site with KLVFFA; b) computational screening strategy.

[0015] Figures 5A-5D provide graphs and images a) single-dose inhibitor screening; b) selection of best inhibitor, fluspirilene, and its dose-dependent LilrB2 inhibition c) Ki and IC50 values; d) predicted binding mode of fluspirilene with other ApRs.

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SUBSTITUTE SHEET (RULE 26) [0016] Figures 6A-6C provide graphs and images showing Fluspirilene inhibits A|342 binding, a) fluorescence images of mouse primary neurons treated with 500 nM FITC-A (green) and 10 .M fluspirilene; b) quantification of FITC-AP42 binding; (**P < 0.005, ANOVA test) c) Quantification of TUNEL cell viability assays (***P<0.0005 two-sided t-tests).

[0017] Figures 7A-7E provide graphs and images showing Flavonoid compounds work as tau inhibitors, a) flavonoid compound library screening and tau biosensor cell testing; b) singledose inhibitor screening; c) fluorescent microscopy images of tau biosensor cells; d) IC50 of tau aggregation and CC50 against human cells.

[0018] Figures 8A-8C provide graphs and images showing Flavonoids disassemble existing tau aggregates, a) Design of tau biosensor cell-based tau disassembly test; b) dose-dependent disassembly results (DC50); c) Myricetin ICV administration to 10 months AD mice and p-tau level immunohistochemical analysis images.

[0019] Figures 9A-9C provide graphs and images showing combination treatment and analysis of tau levels in mice, a) Schematic of CATT mice injection; quantification and analysis of total tau and p-tau (serine 202) levels in 3xTG AD mice (b) hippocampus and (c) Cortex using Western blot (Wes) analysis including stats.

[0020] Figures 10 A- 10C provide a schematic image and graphs showing improvement of Cognitive Function in Animals Following Administration of Tau-Inhibitory Plant Composition in Barnes Maze Test, a) Timeline of in vivo study, including weekly behavioral experiments and brain sample analysis, b) Results and quantification of maze escape time in wild-type control mice at different birth stages following administration of plant extract, c) Improved maze escape time results and quantification in early and late-stage Alzheimer’s disease model mice following administration of plant extract.

DETAILED DESCRIPTION OF THE INVENTION

[0021] AD is the consequence of neuronal death and brain atrophy associated with the aggregation of tau protein into fibrils. Thus, a combined therapy designed to limit A[ - independent tau aggregation, direct disaggregation of formed tau fibrils, limit cell-to-cell spreading, and inhibit the AP-dependent tau aggregation pathway is a promising therapeutic approach to treating AD. See Shin et al., Alzheimers Res Then, 11( 1) :86 (2019). Given recent

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SUBSTITUTE SHEET (RULE 26) findings, the inventors developed a combination anti-tau therapy (CATT) designed to efficiently target both the Ap-dependent and independent mechanisms that can promote the formation of pathological tan tangles. This combination therapy will comprise repurposed drugs that are demonstrated as LilrB2 inhibitors (A[3R inhibitors) and validated plant-based flavonols (tau inhibitors).

[0022] By inhibiting LilrB2, the accumulation of A Os in neurons can be reduced. However, this alone will not completely inhibit the effects of ApO, since other APR pathways still exist. For this reason, the inventors have combined LilrB2 inhibition with direct tau targeting using plant-based flavonols. Their results demonstrate that the combination of both therapeutic avenues (tau inhibitor + APR inhibitor) provides an effective synergistic approach to decreasing tau aggregation (Fig. 1) and significantly impeding the progression of AD and associated cognitive decline.

Definitions

[0023] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0024] As used herein, the term "organic group" is used to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In the context of the present invention, suitable organic groups for the compounds of this invention are those that do not interfere with the neuromodulating activity of the compounds. In the context of the present invention, the term "aliphatic group" means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.

[0025] As used herein, the terms "alkyl", "alkenyl", and the prefix "alk-" are inclusive of straight chain groups and branched chain groups. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. In some embodiments, these groups have a total of at most 10 carbon atoms, at most 8 carbon atoms, at most 6 carbon atoms, or at most 4 carbon atoms. Alkyl groups including 4 or fewer carbon atoms can also be referred to as lower alkyl groups. Alkyl groups can also be referred

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SUBSTITUTE SHEET (RULE 26) to by the number of carbon atoms that they include (i.e., Ci - C4 alkyl groups are alky groups including 1-4 carbon atoms).

[0026] The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may be substituted or unsubstituted.

[0027] Unless otherwise indicated, the term "heteroatom" refers to the atoms 0, S, or N. The term "heteroaryl" includes aromatic rings or ring systems that contain at least one ring heteroatom (e.g., O, S, N). In some embodiments, the term "heteroaryl" includes a ring or ring system that contains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O, S, and/or N as the heteroatoms. Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl, oxadiazolyl, thiadiazolyl, and so on.

[0028] The invention is inclusive of the compounds described herein in any of their pharmaceutically acceptable forms, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, and the like. In particular, if a compound is optically active, the invention specifically includes each of the compound's enantiomers as well as racemic mixtures of the enantiomers. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated or not (although at times, "salts" are explicitly stated).

[0029] As used herein, a subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age. Thus, adult, juvenile, and newborn subjects are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject afflicted with a disease or disorder (e.g. Alzheimer’s disease). The term patient or subject includes human and veterinary subjects.

[0030] Treat", "treating", and "treatment", etc., as used herein, refer to any action decreasing the rate of aging of a subject or providing a benefit to a subject having a neurodegenerative

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SUBSTITUTE SHEET (RULE 26) disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, etc.

[0031] As used herein, the term “prevention” includes either preventing or decreasing the risk of developing a neurodegenerative disease or disorder. This includes prophylactic treatment of those having an enhanced risk of developing Alzheimer’s disease. An elevated risk represents an above-average risk that a subject will develop Alzheimer’s disease, which can be determined, for example, through family history or the detection of genes causing a predisposition to develop a neurodegenerative disease or disorder. A subject can also have an increased risk of developing Alzheimer’s disease as a result of injury, exposure to toxins, or infection.

[0032] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0033] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. The therapeutically effective amount may be administered in one or more doses. An effective amount, on the other hand, is an amount sufficient to provide a significant chemical effect.

Methods of Treating or Preventing a Tau Pathology

[0034] In one aspect, the present invention provides a method for treating or preventing a tau pathology in a subject in need thereof. The method includes administering a therapeutically effective amount of an A0R inhibitor in combination with a quercetin analog to the subject.

[0035] Tau pathologies are diseases and disorders resulting from the accumulation of misfolded tau protein in a subject. Misfolded tau may be present in brains of individuals suffering from AD or suspected of having AD, or other tauopathies that, like AD, regard misfolding in the presence of both 4R and 3R tau isoforms. Misfolded tau may also be present in diseases that regard misfolding of primarily 4R tau isoforms, such as progressive supranuclear palsy (PSP), tau-dependent frontotemporal dementia (FTD), corticobasal

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SUBSTITUTE SHEET (RULE 26) degeneration (CBD), mild cognitive impairment (MCI), argyrophilic grain disease (AgD), and the like.

[0036] In some embodiments, the tan pathology is Alzheimer’s disease. Alzheimer's disease (AD) is a chronic neurodegenerative disease that results in the loss of neurons and synapses in the cerebral cortex and certain subcortical structures, resulting in gross atrophy of the temporal lobe, parietal lobe, and parts of the frontal cortex and cingulate gyrus. Wenk G., The Journal of Clinical Psychiatry. 64 Suppl 9: 7-10 (2003). Alzheimer's disease is usually diagnosed based on the person's medical history, history from relatives, and behavioral observations. The presence of characteristic neurological and neuropsychological features and the absence of alternative conditions is supportive. Advanced medical imaging with computed tomography (CT) or magnetic resonance imaging (MRI), and with single-photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to help exclude other cerebral pathology or subtypes of dementia.

[0037] In some embodiments, the tau pathology is treated. A tau pathology is typically treated in a subject that has been diagnosed as having a tau pathology. In further embodiments, the tau pathology is prevented. A tau pathology is typically prevented, or its likelihood of occurrence decreased, by administration of the APR inhibitor and the quercetin analog in a subject who is at an increased risk of developing a tau pathology.

[0038] The method includes administering a therapeutically effective amount of an amyloidbeta receptor (APR) inhibitor in combination with a quercetin analog to the subject. Examples of suitable APR inhibitors and quercetin analogs are shown in Figure 1. The method includes administration of an APR inhibitor in combination with a quercetin analog. Use in combination, as defined herein, refers to administration of the APR inhibitor and the quercetin analog close enough together in time that they exhibit an additive or synergistic therapeutic effect. Combined administration includes administration of the APR inhibitor before administration of the quercetin analog, administration of the quercetin analog before the APR inhibitor, and administration of the APR inhibitor and the quercetin analog at the same time, either as part of the same formulation, or as separately formulated compounds.

[0039] Several different receptor proteins have been identified that bind monomeric, oligomeric, or fibrillar forms of A . See Smith et al., J Biol Chem., 294(15):6042-6053 (2019) and Jarosz-Griffiths et al., J Biol Chem., 291(7):3174-83 (2016). In some embodiments, the

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SUBSTITUTE SHEET (RULE 26) A^R is LilrB2, and the APR inhibitor is an LilrB2 inhibitor. Examples of LilrB2 inhibitors include miconazole, sertaconazole nitrate, fluspirilene, CDPPB, ML169, ALI6, and TAK-715. See Cao et al., Nat Chem.,10(12):1213-1221 (2018). A variety of APR inhibitors have been developed which inhibit binding to these receptors. Examples of the APR inhibitors include TAK-715 and fluspirilene. In some embodiments, the APR inhibitor is fluspirilene. As described herein, APR inhibitors can be identified by testing compounds for their based on their docking score (energy), expected toxicity, and reported BBB permeability, and through quantitative immunoprecipitation assays with the LilrB2 D1D2 binding domain and oligomeric A 42.

[0040] The method also includes the administration of a quercetin analog. A number of quercetin analogs are known to those skilled in the art. See Septembre-Malaterre et al., Phytomed Plus., 2(1), 100220 (2022), and Pavlovic et al., Anticancer Agents Med Chem., 22(7): 1407-1413 (2022), the disclosures of which are incorporated herein by reference. Analogs typically have structurally similar nuclei but will differ in a particular chemical moiety. For example, alcohols and esters represent analogs, differing in the presence of a hydrogen and an alkyl group. The structure of quercetin is shown below:

[0041 ] Quercetin and structurally similar natural products maintain an unsaturated bond at C2- C3 and a ketone at C4 of the C-ring and are a part of the flavone subclass of flavonoids. Examples of quercetin analogs include kaempferol, chrysin, apigenin, baicalein, negletein, wogonin, nobiletin, myricetin, quercetagetin, and gossypetin. In some embodiments, the quercetin analog is selected from the group consisting of myricetin, quercetagetin, and gossypetin. Quercetin analogs can be tested for activity by testing their ability inhibit brain- derived tau protein using the tau biosensor cell assay, as described herein.

Anti-tau pathology Compositions

[0042] A further aspect of the invention provides an anti-tau pathology composition, comprising a therapeutically effective amount of an A[)R inhibitor in combination with a

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SUBSTITUTE SHEET (RULE 26) quercetin analog. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition consists of an A0R inhibitor, a quercetin analog, and a pharmaceutically acceptable carrier. In further embodiments, the composition is formulated for administration by injection.

[0043] The APR inhibitor and quercetin analog included in the composition can be any of the APR inhibitors and quercetin analogs described herein. In some embodiments, the APR inhibitor is an LIlrB2 inhibitor. In further embodiments, the APR inhibitor is fluspirilene. In yet further embodiments, the quercetin analog is selected from the group consisting of myricetin, quercetagetin, and gossypetin.

[0044] The amount of APR inhibitor and quercetin analog included in the composition can vary, depending on the particular condition being treated and the activity of the specific compounds being used. In some embodiments, the amount of APR inhibitor is substantially greater than the amount of quercetin analog. In some embodiments, the amount of APR inhibitor is substantially lower than the amount of quercetin analog. In some embodiments, the amounts of the APR inhibitor and the quercetin analog are about the same.

Formulation and Administration

[0045] In some embodiments, the present invention provides a method for administering an ApR inhibitor and a quercetin analog (i.e., the active compounds) in a pharmaceutical composition. In some embodiments, the method described herein consists of administering a therapeutically effective amount of an APR inhibitor in combination with a quercetin analog to the subject and a pharmaceutically acceptable carrier.

[0046] Examples of pharmaceutical compositions include those for oral, intravenous, intramuscular, subcutaneous, or intraperitoneal administration, or any other route known to those skilled in the art, and generally involves providing a neuromodulating compound formulated together with a pharmaceutically acceptable carrier.

[0047] When preparing the active compounds for oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active compounds. Examples of such dosage units are capsules,

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SUBSTITUTE SHEET (RULE 26) tablets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethyl-cellulose; and with lubricants such as talc or magnesium stearate. The active compound may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier.

[0048] In some embodiments, the A0R inhibitor and the quercetin analog are administered by injection. For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, the active compounds may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredients in water containing physiologically compatible substances such as sodium chloride, glycine, and the like and having a buffered pH compatible with physiological conditions to produce an aqueous solution and rendering said solution sterile. The formulations may be present in unit or multi-dose containers such as sealed ampoules or vials.

[0049] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound, which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in nonaqueous solvents, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol.

[0050] The dosage form and amount can be readily established by reference to known treatment or prophylactic regiments. The amount of active compound(s) that is administered and the dosage regimen for treating a tau pathology using these compounds depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, the location of the unwanted proliferating cells, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime or therapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual. The pharmaceutical compositions

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SUBSTITUTE SHEET (RULE 26) may contain active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50 mg/kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

[0051] The active compounds can also be provided as pharmaceutically acceptable salts. The phrase “pharmaceutically acceptable salts” connotes salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2- hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, y-hydroxybutyric, galactaric, and galacturonic acids. Suitable pharmaceutically acceptable base addition salts of the compounds described herein include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Alternatively, organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine may be used form base addition salts of the compounds described herein. All of these salts may be prepared by conventional means from the corresponding compounds described herein by reacting, for example, the appropriate acid or base with the compound.

[0052] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

[0053] The work described in these examples will (i) broaden our understanding of how plant flavonol compounds are able to bind to tau and force its disassembly, providing additional key

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SUBSTITUTE SHEET (RULE 26) information for future tau-targeted drug development; (ii) provide additional evidence for the use of "old" drugs as LilrB2 antagonists in AD treatment; and (iii) demonstrate the feasibility of a combination to inhibit tau fibrillization and retard AD progression.

Example 1: Amyloid- B oligomers promote the uptake of tau fibril seeds, potentiating intracellular tau aggregation

[0054] Repeated failure of drug candidates targeting AD in clinical trials likely stems from a lack of understanding of the molecular mechanisms underlying AD pathogenesis. Recent research has highlighted synergistic interactions between aggregated A[3 and tau proteins in AD, but the molecular details of how these interactions drive AD pathology remain elusive and speculative. We have demonstrated that the presence of ApOs potentiated the formation of tau fibrils within tau biosensor cells. This was demonstrated using a FRET-based tau biosensor cell assay, where various A units (monomer, oligomer, or fibril) could be applied. It was observed that ApOs, but not monomers or fibrils, “primed” the cells and made them susceptible to tau seeding (Fig. 3a, b). We further examined this AP-induced tau aggregation phenomenon in human neuroblastoma cells and mice primary neurons (Fig. 3 c, d). There, we confirmed that ApOs promoted tau aggregation, linking Ap toxicity to tau spreading in cells and connecting a previously missing link in understanding AD pathology (Fig. 3e).

Example 2; Discovery of an A receptor, LilrB2, inhibitor, Fluspirilene, using a drug repurposing strategy

[0055] With the observation that ApOs could potentiate the formation of neurotoxic tau tangles, we sought to determine whether any drugs could be repurposed as ApRIs. We obtained the structural information of the LiIrB2 binding site and screened a large library of compounds (2,700 approved drugs, 9,600 drugs in clinical trials, 46,000 drug candidates tested in animals or humans, and 58,000 natural products) for potential inhibitors (Fig. 4).

[0056] In doing so, we identified 14 possible drug candidates (ApRIs 1-14) based on their docking score (energy), expected toxicity, and reported BBB permeability. We tested the inhibitory ability of all candidates through quantitative immunoprecipitation assays with the LilrB2 D1D2 binding domain (Fig. 4a) and oligomeric AP42 (Fig. 5a). In a single-dose test, 10 out of the 14 candidates exhibited inhibition of LilrB2, with the top 6 candidates (ApRI #4, 5, 6, 7, 9,10) selected for further dose-dependent studies (Fig. 5b, c). Of these six candidates,

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SUBSTITUTE SHEET (RULE 26) A0RI #6 (fluspirilene) and ApRI #10 (TAK-715) were found to be the best potential inhibitors, exhibiting both nanomolar inhibition constants (Ki) and low micromolar-nanomolar IC50 values. Interestingly, both of these drug candidates have been computationally predicted to bind to other major A^Rs, including Prion and EphB2 (Fig. 5d).

[0057] We further validated the APR (LiIrB2) inhibitory effect of putative inhibitors on mouse primary neurons. Cells from cortices dissected at embryonic day 15 were dispersed and cultured for 14 days in-vitro. Cells were then treated with 500 nM FITC-AP42 to assess Ap binding. We found that cells pretreated with 10 pM ApRI #6 (fluspirilene) bound 39.0 ± 20.5% of FITC-AP42 compared to cells pretreated with the same amount of DMSO (Fig. 6a), indicating that ApRI #6 inhibits the binding of Ap to neurons. The observation that ApRI #6 does not fully inhibit Ap binding, even at a higher dose (50 pM, Fig. 6b), indicates that there are ApR other than LilrB2 on the neuronal cell surface. In this manner, cells treated with fluspirilene did not achieve complete elimination of Ap binding, but we further discovered that ApRI #6 is sufficient to inhibit AP-induced cellular toxicity in primary neurons (Fig. 6c).

[0058] Based on these results, we have selected ApRI #6 fluspirilene and #10 TAK-715 as primary compounds for a combination therapy targeting key AP-dependent pathways. Fluspirilene is an antipsychotic agent that was first approved in the 1970’ s Canada, EU and is still an approved therapy in some European countries and the US (marketed as IMAP). It has also been recently investigated as a depot agent for long-term maintenance therapy for schizophrenia as well as mentioned in a German report, suggesting its use as a potential treatment for AD patients. These reports suggest that this is a well-characterized drug that can be safely utilized in the clinic. Additionally, the fact that this is a CNS-targeting drug indicates efficient BBB penetration. The second lead compound, TAK-715, was previously developed by Takeda Pharmaceuticals as an anti-rheumatoid arthritis agent. A phase II clinical trial was conducted in 2005; however, no results have been published. Importantly, this agent is generally insoluble in water, indicating high lipophilicity and may therefore also exhibit BBB permeability. Given the fact that both compounds have been previously investigated and their safety has been already demonstrated, the choice of these as possible lead compounds aligns with their intended use as A[3R (LiIrB2) targeting agents in our combination therapy.

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SUBSTITUTE SHEET (RULE 26) Example 3: Discovery of a flavonoid compound as a tau aggregation inhibitor

[0059] Following our previous work on A[3RIs, we began to search for direct tau inhibitors and tau disassemblers. Such compounds would act directly on tau with the goal of further reducing the number of aggregated tau fibrils since A0R inhibition alone cannot guarantee the total blockade of A0O uptake. Based on numerous previous reports on tau inhibition by plant- derived polyphenols or flavonoids as well as our own in-silico compound library screening (541 initial compounds), we tested the aggregation-inhibitory abilities of the final twenty compounds against in a HEK293T tau biosensor cell assay (Fig. 7a). Unlike previous studies, which typically used recombinant tau (tau 40, RD), our tau biosensor cell assay utilizes 3xTG- derived mouse Hippocampus (HC) and pre-frontal cortex (PFC) tau as seeds. This allowed us to confidently assess whether the brain samples from 3xTG AD mice were activated at the cellular level as a seed, along with determining whether the screened flavonoid candidates could inhibit the target mice’s brain-derived tau protein.

[0060] Impressively, our repeated single-dose testing results identified three flavonoid compounds (Fig 7b) that strongly inhibit tau aggregation. Although these compounds possess relatively similar structures but exhibit significantly different activities in terms of AD brain- derived tau aggregation inhibition. For example, the well-known flavonoid quercetin (#1) shares a related structure but exhibits limited activity. Tau biosensor cells were treated with the three top flavonoid candidates at 10 different concentrations (0.01-25 pM) along with AD mice-brain-derived tau seeds for 24 hours. The results indicated that myricetin (#5), quercetagetin (#6), and gossypetin (#11) exhibited ICso values of 0.91 pM, 1.21 pM, and 0.83 pM, respectively (Fig. 7c, d, e). Furthermore, we validated our final three compounds using tau fibrils obtained from AD human brain (HB) tissue. Impressively, this reaffirmed our findings, with myricetin (#5), quercetagetin (#6), and gossypetin (#11) exhibiting IC50 values of 2.87 pM, 1.1 pM, and 0.22 pM, respectively. (Fig 7e) Both sets of results agree with previous tau-RD studies (myricetin 1.2 pM and gossypetin 2 pM). Importantly, these results strongly support the use of polyphenol flavonoids as tau inhibitors given that they are low micromolar to nanomolar range inhibitors and that related compounds have also been reported to be capable of disassembling tau fibrils at higher concentrations.

[0061] The ability to disassemble existing filaments may be an important factor for a potential tau-targeting agent as this could potentially lead to a reversal of the disease state. To determine

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SUBSTITUTE SHEET (RULE 26) whether compounds myricetin (#5), quercetagetin (#6), and gossypetin (#6) could disassemble tau aggregates, we developed a procedure in which tau biosensor cells were pre-treated with HC/PFC tau seeds prior to inhibitor treatment, allowing for maximum tau aggregation to occur by pre-treatment. Then, after 24 hrs of seed incubation, inhibitors were treated, and the cells were incubated for the following 72 hrs (Fig. 8a).

[0062] In doing so, we were able to observe a dose-dependent decrease in tau levels with approximate half-maximal disassembly concentration (DC50) values of 7.5 pM, 14 pM, and 9.4 pM for myricetin (#5), quercetagetin (#6), and gossypetin (#6), respectively (Fig 7b). If such concentrations could be obtained in-vivo, then this would potentially allow for tau clearing as well as tau inhibition.

[0063] To determine how well mice could tolerate such high concentrations (180 pg/ml) of these flavonoids, we administered a 0.5 mM solution of flavonoids directly into the brains of 10-month-old 3xtg mice (3 pl) using intracerebroventricular (ICV) injection. All 10 administered mice exhibited no signs of distress or discomfort, and the administered dose was well-tolerated. After 48 hr, the mice were euthanized, and the brains were sectioned and stained for phosphorylated tau (ptau serine 202; Fig. 8b). Subsequently, we observed through immunofluorescence imaging that the brains of 3xTG AD mice treated with myricetin (#5) for 48 h exhibited lower ptau levels in the cortex and HC regions compared to the control, supporting the hypothesis that the flavonoids may serve a dual role as both tau inhibiting and tau clearing agents (Fig. 8c). Alongside minimal toxicity to mouse brains, these inhibitory functions can potentially lead to the future application of flavonoids in combination with A0R inhibitors to limit tau potentiation, inhibit tau aggregation, and disassemble pre-existing tau fibrils in the brains of AD patients. These studies are further supported by reports of BBB penetration by these polyphenols, even when administered orally.

Example 4: Efficacy test of combination therapy in animal model

[0064] The onset of AD pathology in 3xTg-AD mice is known to be observed within 3 to 4 months. In contrast to recent animal experiments where we employed severely afflicted 3xTg- AD mice aged over 10 months to observe a dramatic inhibitory effect on ptau levels after 48 hours of treatment with flavonoid candidate compounds (myricetin #5, Fig. 8c), in our next efficacy test of the tau inhibitors alone and the combination therapy in-vivo, we employed 3- month-old 3xTg-AD mouse models (Fig. 9a).

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SUBSTITUTE SHEET (RULE 26) [0065] Alternatively, we also examined the ability of our combination therapy treatment to control and regulate the onset of early-stage AD pathology. A total of 16 three- month-old female 3xTg-AD mice were divided into four groups, each consisting of four mice. These groups received separate treatments: a control group with phosphate-buffered saline (PBS) injection, an A0R inhibitor (fluspirilene), a tau inhibitor (#5, myricetin), and a combination therapy group with both tau inhibitor (#5 myricetin) and A[>RI (fluspirilene). The tau inhibitor was administered at a dosage of 1 mN (ICV), which had previously passed assessments for both its efficacy and toxicity in animal testing. Additionally, fluspirilene was administered via intramuscular (IM) injection in a single or 1 :1 dose range with the tau inhibitor, within the standard maintenance dosage range of 1 to 10 mg weekly. Following administration, each group of mice would undergo a recovery period of three weeks, during which they were expected to exhibit distinct AD pathological progression patterns. After the three-week period, mice in each group underwent euthanasia, and total tau/p-tau levels in brain tissues were measured (Fig. 9).

[0066] The dependent variables we assessed: were phosphorylated tau (ptau serine 202), and total tau (tau5), and computed a new variable of what we commonly use in the field as a “pathological tau load” — a ratio of ptau to total tau against two brain structures (hippocampus and cortex). The overall statistical results of the pilot data, with n of 4/per group, may be somewhat limited in highlighting a significant inhibitory effect. However, the patterns exhibited in the graphed data adequately support our hypothesis. For Figure 9b (hippocampus), we see a trend in the data showing that both the Tau inhibitor and CATT groups have reduced p-tau relative to the vehicle group. For Figure 9c (cortex), we also see a clear decrease in p-tau level in the tau inhibitor and CATT groups relative to the vehicle group. Examining the reduction in pathological tau loads for each group (Fig. 9b, c, right panel), the difference between the tau inhibitor and CATT may not be very pronounced at this stage. However, the proposed increase in animal numbers and additional rounds of IV tau inhibitor administration may significantly increase these apparent differences. Additionally, A|3RI/taii inhibitor monotherapy appears to have a similar effect on tau pathology, but the addition of A[3 R I as a combination therapy will be crucial as part of a real therapy. Flavonoids may disassemble and inhibit tau fibrilization, However, concurrently, the ongoing uptake and interplay of ApOs will continually enhance the formation of new tau fibrils. Therefore, a combination of both agents is necessary in order to have a meaningful and comprehensive effect on AD tau pathology.

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SUBSTITUTE SHEET (RULE 26) [0067] Behavioral test using the Barnes Maze assess both reference and working memory, making it an ideal memory test for rodents. In this test, visual cues are placed within the testing room, and the maze is illuminated. The participating rats were first habituated to the escape tunnel and the center of the apparatus (San Diego Instruments; diameter 36 inches, 20 holes with 2-inch diameters). During the acquisition phase (10 days), each rat was given two trials per day, where they were placed in the center of the maze and trained to explore and locate the escape tunnel. Three days after acquisition, a probe trial was conducted by placing the animals in the center of the apparatus with the escape tunnel removed. Latency, distance traveled, identified target holes, and identified incorrect holes were recorded.

[0068] Our results from the Barnes Maze behavioral test demonstrated a statistically significant reduction in the time it took for the tested mice to escape the maze, with increased activity levels observed in both early and advanced-stage dementia model mice. This indicates a significant and effective improvement in memory and cognitive functions impaired by Alzheimer's tau aggregation and tau fiber accumulation, along with a reduction in phosphorylated tau levels (see Figures 10a- 10c). Subsequent analysis of brain tissue from the dementia model mice confirmed the progression of improvements in Alzheimer's disease pathology.

[0069] Collectively, our data support the notion that with appropriate optimization, the planned combination therapy may: 1) effectively halt tau aggregation and significantly impede the progression of AD with tau inhibitors (flavonoids), and 2) demonstrate strong efficacy in inhibiting the pathological mechanisms pathway of tau enhanced by A[3, with the additional administration of A^R inhibitor (fluspirilene).

[0070] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. In particular, while various theories are presented describing possible mechanisms through with the compounds are effective, the compounds are effective regardless of the particular mechanism employed and the inventors are therefore not bound by theories described herein. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

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SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS What is claimed is:

1. A method for treating or preventing a tau pathology in a subject in need thereof, comprising administering a therapeutically effective amount of an ApR inhibitor in combination with a quercetin analog to the subject.

2. The method of claim 1, wherein the A R inhibitor is an LIlrB2 inhibitor.

3. The method of claim 1, wherein the APR inhibitor is fluspirilene.

4. The method of claim 1, wherein the quercetin analog is selected from the group consisting of myricetin, quercetagetin, and gossypetin.

5. The method of claim 1, wherein the method consists of administering a therapeutically effective amount of an APR inhibitors in combination with a quercetin analog to the subject and a pharmaceutically acceptable carrier.

6. The method of claim 1, wherein the tau pathology is treated.

7. The method of claim 1, wherein the tau pathology is prevented.

8. The method of claim 1, wherein the tau pathology is Alzheimer’s disease.

9. The method of claim 1, wherein the subject is a human subject.

10. The method of claim 1, wherein the APR inhibitor and the quercetin analog are administered by injection.

11. An anti-tau pathology composition, comprising a therapeutically effective amount of an APR inhibitor in combination with a quercetin analog.

12. The anti-tau pathology composition of claim 11, wherein the composition further comprises a pharmaceutically acceptable carrier.

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13. The anti-tau pathology composition of claim 11 , wherein the ApR inhibitor is an LIlrB2 inhibitor.

14. The anti-tau pathology composition of claim 11, wherein the APR inhibitor is fluspirilene.

15. The anti-tau pathology composition of claim 11, wherein the quercetin analog is selected from the group consisting of myricetin, quercetagetin, and gossypetin.

16. The anti-tau pathology composition of claim 11 , wherein the composition is formulated for administration by injection.

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