METHODS AND COMPOSITIONS RELATED TO HEPARINOIDS

TECHNICAL FIELD

[0001] Embodiments of the disclosure relate generally to compositions comprising novel heparinoid-related compounds and to methods of making the same.

BACKGROUND OF THE INVENTION

[0002] Tauopathies are a class of neurodegenerative disorders characterized by neuronal and/or glial inclusions composed of the microtubule-binding protein, tau. Several lines of evidence suggest tau aggregation is central to the neurodegenerative process in tauopathies. Tangles are formed by hyperphosphorylation of a microtubule-associated protein known as tau, causing the protein to dissociate from microtubules and form aggregates in an insoluble form. Such aggregations of hyperphosphorylated tau protein may also be referred to as paired helical filaments. Tauopathies include an array of disorders: primary age-related tauopathy (PART)/Neurofibrillary tangle (NFT) -predominant senile dementia, (with NFTs similar to Alzheimer’s Disease (AD), but without plaques,) chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17, Fytico-bodig disease (Parkinson- dementia complex of Guam), ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[0003] In AD, pathological tau aggregation spreads progressively throughout the brain, possibly along existing neural networks. Studies have demonstrated that tau aggregation is facilitated by transcellular propagation via heparan sulfate proteoglycans (HSPGs). AD is the most common cause of dementia and is an increasing public health problem. It is currently estimated to afflict 5 million people in the United States, with an expected increase to 13 million by the year 2050. Alzheimer's Disease leads to loss of memory, cognitive function, and ultimately loss of independence. It takes a heavy personal and financial toll on the patient and the family. Because of the severity and increasing prevalence of the AD and other neurodegenerative diseases associated with aggregation of tau in the population, it is urgent that better treatments and detection methods be developed.

[0004] Recent findings have revealed that other amyloid protein aggregates, and not just tau aggregates, are also associated with pathophysiologies that contribute to disease via transcellular propagation. Such diseases include cancer and metabolic disease (including diabetes). For example, in the case of type II diabetes, the protein islet-associated polypeptide (IAPP) may mediate spread of pathology within the cells of the pancreas. In the case of cancer, oncogenic protein p53 also propagates pathology via binding to HSPGs.

[0005] What is needed therefore, are novel compounds, compositions and methods of making the same that effectively prevent, reduce and/or inhibit the ability of aggregates, such as protein aggregates, to effectuate disease and disorder via transcellular propagation. In particular, what is needed are effective compounds and compositions that reduce, prevent and/or inhibit the binding of protein aggregates, such as amyloid structures including tau aggregates, from binding to receptors such as HSPGs. Such novel compounds, compositions and methods of making the same should have minimal side-effects, and function effectively to reduce or eliminate the uptake and amplification of aggregates, such as those derived from tau.

SUMMARY OF THE INVENTION

[0006] Provided herein are novel compounds, compositions and methods of making the same that effectively prevent, reduce and inhibit the ability of aggregates, such as tau amyloids, to effectuate disease and disorder via transcellular propagation. In particular, provided herein are novel heparinoid-like compounds and methods for making the same. The novel compounds block macromolecular aggregate uptake and amplification, including tau aggregate cell uptake and amplification, and lack side-effects such as anticoagulation activity.

[0007] Tau aggregation underlies neurodegeneration in numerous tauopathies including Alzheimer’s disease (AD). The inventors and others have proposed that transcellular propagation of AD pathology is mediated by tau prions, which are ordered assemblies that faithfully replicate to cause biological effects. The model predicts the release of aggregates from a first order cell with subsequent uptake in a second order cell. The assemblies then serve as templates for their own replication, a process termed “seeding.” The inventors have previously observed that heparan sulfate proteoglycans (HSPGs) on the cell surface mediate the cellular uptake of tau aggregates. This interaction is blocked by heparin, a sulfated glycosaminoglycan. Indeed, heparin-like molecules, or heparinoids, have previously been proposed as therapies for PrP prion disorders. However, heparin is not ideal to treat chronic neurodegeneration, as it is difficult to synthesize in defined sizes, is highly polyanionic, and is a powerful anticoagulant. In an effort to meet the unmet need of compounds that effectively bind HSPGs without unwanted side-effects, the inventors herein have generated novel oligosaccharides that bind tau and block its cell uptake and seeding, without anticoagulation activity. The novel oligosaccharides described herein comprise compounds synthesized from pentasaccharide units that bind tau with low nanomolar affinity, inhibit cellular tau uptake and seeding, similar to standard porcine heparin, but do not inhibit coagulation. Use of the novel oligosaccharides described herein is contemplated for the development of therapeutic heparanoids.

[0008] The common minimal connection between Alzheimer's Disease and all the tauopathies is the aggregation state of tau. Under all these diseased conditions, monomeric tau is known to be converted into polymeric ordered fibrils. Neurofibrillary tangles (NFTs), which are comprised of fibrillar tau aggregates, are a neuropathological hallmark of tauopathies. It has been discovered that spreading of tau pathology in the brain may be caused by a form of tau aggregate released from a "donor" cell entering a second "recipient" cell, and inducing further misfolding and aggregation of tau in the recipient cell via direct protein-protein contact. The specific form of tau aggregate which facilitates this cell-to-cell spread of tau aggregates is referred to as "tau seeds" and the activity may be referred to herein as "seeding activity", since this form of tau aggregate seeds or nucleates tau aggregation in the cell it enters (i.e. the "recipient cell").

[0009] Tau can exist in both a monomeric form and in different aggregated forms. As used herein, the term "tau aggregate" refers to a molecular complex that comprises two or more tau monomers. It is contemplated that a tau aggregate may comprise a nearly unlimited number of monomers bound together. For example, a tau aggregate may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more tau monomers. Alternatively, a tau aggregate may comprise 20, 30, 40, 50, 60, 70, 80, 90, 100 or more tau monomers. A tau aggregate may also comprise 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more tau monomers. The terms "fibrillar tau aggregate" and "tau fibril" refer to forms of tau aggregates, and these terms are used interchangeably herein. A fibrillar tau aggregate is a polymeric, ordered fiber comprising tau. Tau fibrils are generally not soluble, but shorter assemblies, or oligomers, can be soluble. Tau aggregate also refers to soluble tau oligomers and protofibrils, which may act as intermediates during tau aggregation. Also included in the definition of tau aggregate is the term "tau seed", which refers to a tau aggregate that is capable of nucleating or "seeding" intracellular tau aggregation when internalized by a cell, or when exposed to monomeric tau in vitro.

[00010] As discussed above, it is contemplated that transcellular propagation of tau assemblies may mediate progression of human neurodegenerative diseases. According to this model, aggregates released from one cell gain entry to a second order cell and act as templates for their own replication in the manner of prions. Cell binding is thus a potentially critical point in the progression of disease. HSPGs are transmembrane and glycolipid-anchored proteins that are decorated with sulfated glycosaminoglycans. HSPGs directly bind tau aggregates and mediate their cellular uptake and subsequent intracellular seeding activity (1,2). Heparan sulfate (HS) is a linear glycosaminoglycan (GAG) expressed in all known eukaryotic cells (3- 5). Its basic disaccharide unit consists of uronic acid (iduronic or glucuronic acid) and glucosamine with potential sulfate moieties at N-, C2-, C3-, and C6-positions and variable N- acylation or -sulfation. During synthesis of HSPGs in the Golgi apparatus, the HS chain is covalently linked to core proteins (3-5). As surface receptors or co-receptors, HSPGs mediate processes such as embryogenesis, cell migration and hemocoagulation (3,4), and are thus potentially powerful therapeutic targets. The interaction between HSPGs and protein binding partners can be highly specific, and depends on the charge, modification, and length of the GAG chain (3,4).

[00011] Heparin is a polydisperse GAG, typically 3-30kDa, with a structure similar to heparan sulfate (HS). It is therefore frequently used to assess the effects of HS in biological systems (3,4,6). Importantly, heparin is only secreted by mast cells, and differs from HS in a higher sulfate and charge density, shorter chain length, and a higher content of iduronic acid (4,7). Heparin is a potent anticoagulant that binds and activates antithrombin via a pentasaccharide sequence (8-10).

[00012] Heparin and heparin derivatives are thought to be useful for potentially inhibiting pathology in neurodegenerative disorders. The inventors have observed that heparin and heparin- like molecules (heparinoids) competitively inhibit cellular tau uptake (1,2) and decrease tau-induced cell toxicity (11). Similarly, the cellular uptake of prion protein aggregates is mediated by HSPGs and can be blocked by GAG derivatives in vitro (7,12,13), and GAGs have been observed to reduce prion pathology in vivo (14,15). Finally, low molecular weight heparins (LMWH) such as neuroparin and enoxaparin have demonstrated neuroprotective effects in mouse models of Alzheimer’s Disease (AD) (16-20). Taken together, GAGs may be a promising therapeutic tool to inhibit the progression of pathology in tauopathies and other neurodegenerative disorders. A significant challenge with such an approach is that because of its high MW and charge, anticoagulation properties, and the associated risk of hemorrhages, heparin is not suitable for chronic administration to treat neurodegeneration. In an effort to solve these problems, the inventors have engineered and discovered novel low molecular weight heparinoids that inhibit tau uptake and intracellular seeding, and also lack anticoagulation activity.

[00013] Provided herein are novel heparinoid-like compounds, wherein pentasaccharide building blocks with “linkable linker” side chains (A) and N,N-(Disuccinimidyl) suberate (B) are used to obtain a polydisperse compound mixture of oligosaccharides (C) with a MW ranging from 2500 to 8000 Da containing variable numbers of the pentasaccharide building block units. The novel compounds described herein may be utilized in pharmaceutical compositions for therapeutic intervention in central nervous system disorders including, but not limited to, neurodegenerative and/or prion related disorders. Based on a similar principle, the novel compounds described herein may be further utilized to block transcellular propagation of assemblies other than tau aggregates, including for example, those macromolecular aggregates thought to be involved in other diseases and disorders such as cancer and other metabolic diseases.

BRIEF DESCRIPTION OF THE FIGURES [00014] Figure 1 provides, scheme 1, compound 1; commonly named N-l Fondaparinux used for producing heparin-like oligosaccharide molecules.

[00015] Figure 2A provides a synthesis scheme and putative structure of SN7-13 wherein a pentasaccharide building block with “linkable linker” side chains (A) and N,N- (Disuccinimidyl) suberate (B) were used to obtain a polydisperse compound mixture of oligosaccharides (C) with a MW ranging from 2500 to 8000 Da, and containing variable numbers of the pentasaccharide building block units. Figure 2B provides a putative structure of the tetra-pentasaccharide component in SN7-13. SN7-13 is a polydisperse mixture of oligosaccharides and contains oligosaccharides composed of 2-4 pentasaccharide building blocks. Here, the putative linear structure of the tetra-pentasaccharide component, which contains four pentasaccharide units linked by carbon chains is shown.

[00016] Figures 3A and 3B provide graphs showing that SN7-13 binds to tau fibrils with low nanomolar affinity. Bio-layer interferometry (BLI) was used to determine binding of tau monomer to SN7-13 (A) and heparin (B). The figures show representative data of SN7-13 and heparin binding at the indicated concentrations to BLI Superstreptavidin sensors loaded with biotinylated tau monomer. The apparent KD was calculated as the mean of two independent experiments +/- SD. Based on titration of compounds, the apparent KD for tamheparin was 60 +/- 30 pM, and for tau:SN7-13 it was 2 +/- 1 nM. [00017] Figure 4 provides scheme 2 (N-Sulfation of Compound (2)).

[00018] Figure 5 provides scheme 3 (Preparation of Building Block (4)): a) DMF, diFbO,

Reagent 4, 50°C, 6h; b) HC1, MeOH, RT, 18h.

[00019] Figure 6 provides scheme 4 (synthesis of Oligo (pentasaccharide) from Building Block (4).

[00020] Figure 7 provides scheme 5 (preparation of Compound (6)).

[00021] Figure 8 provides scheme 6 (synthesis of Reagent II).

[00022] Figure 9 provides an overlay chromatograms of the reference Enoxaparin and the oligopentasaccharide (5) batch 5: SN7-13.

[00023] Figure 10 provides chromatograms demonstrating the reproducibility of the oligomerization protocol: Overlay chromatograms of oligo (pentasaccharide) 5 (Batches: 1-5: SN5-115, SN7-5, SN7-9, SN7-11, and SN7-13).

[00024] Figure 11 provides chromatograms demonstrating the reproducibility of the oligomerization protocol: Mass spectra of three batches (SN7-5, 9, and 11) and SN5-139. The first three batches were prepared by the procedure described herein and compared with a batch that was prepared by a different procedure.

[00025] Figures 12A and 12B provide graphs demonstrating that SN7-13 inhibits uptake of Tau fibrils and oligomers in HEK293T cells. Recombinant Tau fibrils (A) labeled with Alexa Fluor 647 fluorescent dye were applied to HEK293T cells with increasing doses of heparin or SN7-13. Tau uptake was quantified based on the MFI per cell by flow cytometry. Heparin and SN7-13 dose-dependently decreased cellular uptake in both cell types. For uptake with Tau oligomers (B), Tau 3-, 10-, and 20-mer were purified using SEC, and labeled with Alexa Fluor 647 fluorescent dye. No inhibitor (labeled 0 pg/ml), heparin at 200 pg/ml (H200), or SN7-13 at 20 pg/ml (S20) and 200 pg/ml (S200) was simultaneously applied. Both heparin and SN7- 13 efficiently inhibited uptake ofTau oligomers. For both A and B, each condition was recorded in triplicate. The values in A represent the average of three separate experiments. The values in B represent data from one experiment. The uptake in untreated samples was defined as 100%. The uptake in treated samples is shown relative to the untreated samples. The error bars indicate S.D.

[00026] Figures 13A and 13B provide graphs demonstrating that SN7-13 blocks Tau seeding with Tau fibrils and oligomers. A, HEK293T biosensor cells were exposed to Tau fibril seeds in the presence of heparin or SN7-13. Heparin and SN7-13 efficiently inhibited seeding by recombinant Tau. B, Tau oligomers (3-, 10-, and 20-mer) were purified using SEC and applied to HEK293T biosensor cells. No inhibitor (labeled 0 pg/ml), heparin at 200 pg/ml (H200), or SN7-13 at 20 pg/ml (S20) and 200 pg/ml (S200) was simultaneously applied. Both heparin and SN7-13 efficiently inhibited seeding by Tau oligomers. For both A and B, each condition was recorded in triplicate. The values in A represent the average of three separate experiments. The values in B represent data from one experiment. The seeding in untreated samples was defined as 100%. The seeding in treated samples is shown relative to the untreated samples. The error bars indicate S.D.

[00027] Figure 14 provides a graph demonstrating that SN7-13 inhibits Tau uptake in primary neurons. Recombinant Tau fibrils labeled with Alexa Fluor 647 were applied to mouse hippocampal neurons with increasing doses of heparin or SN7-13. Aggregate uptake was quantified by flow cytometry based on the MFI per cell. Heparin and SN7-13 dose- dependently decreased cellular uptake in both cell types. Each condition was recorded in triplicate, and the values represent the averages of three separate experiments. The uptake in untreated samples was defined as 100%. The uptake in treated samples is shown relative to the untreated samples. The error bars indicate S.D.

[00028] Figure 15 provides a graph demonstrating that SN7-13 blocks Tau seeding with brain lysate. HEK293T biosen-sor cells were used to compare the activities of Heparin and SN7-13 as inhibitors of seeding with brain lysate from transgenic mice expressing 1N4R P301S Tau (33). Heparin potently inhibited seeding with brain lysate. SN7-13 inhibited seeding at higher concentrations. Each condition was tested in triplicate. The values represent the averages of three separate experiments. The seeding activity in untreated samples was defined as 100%. The seeding activity in treated samples is shown relative to the untreated samples. The error bars indicate S.D.

DETAILED DESCRIPTION OF THE INVENTION [00029] The present invention is described herein with reference to particular embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that these features may be used singularly or in any combination based on the requirements and specifications of a given application or design. Embodiments comprising various features may also consist of or consist essentially of those various features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary or explanatory in nature and, thus, variations that do not depart from the essence of the invention are intended to be within the scope of the invention. All prior art references cited herein are incorporated by reference in their entirety. United States Provisional Application Serial Number 62/959,542 filed on January 10, 2020 is also incorporated herein in its entirety.

[00030] Provided herein are novel compounds related to heparinoids. As described below, such novel compounds mimic heparan sulfate proteoglycans (HSPGs) on the surfaces of cells, and effectively reduce, prevent and/or inhibit transcellular propagation of protein aggregates by serving as “false receptors” that inhibit aggregate binding to the cell surface. By way of example, in certain embodiments, transcellular propagation of protein aggregates mediated by amyloid structures binding HSPGs on the cell surface may be reduced, prevented or inhibited by the novel compounds described herein. Binding of aggregates to HSPGs is hypothesized to mediate progression of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis, among others. The inventors herein contemplate that additional diseases outside the central nervous system, such as type II diabetes, cancer, and systemic amyloidosis may utilize similar mechanisms, whereby protein aggregates bind HSPGs to mediate cell entry and intracellular amplification of aggregates. In the case of type II diabetes, the protein islet-associated polypeptide (IAPP) may mediate spread of pathology within the cells of the pancreas. In cancer, the oncogenic protein p53 also forms amyloids (ordered aggregates), and may propagate pathology between cells mediated by binding HSPGs on the cell surface. In this model, a p53 aggregate gains entry to a cell and “corrupts” native p53, disabling its function, and causing oncogenic transformation of the second order cell. In addition, certain viral particles (also macromolecules similar in size to amyloid aggregates) can bind the cell surface via HSPGs, similar to amyloid proteins. The novel heparinoid compounds designed and created by the inventors, including SN7-13 and/or related molecules, bind a variety of ordered amyloids and macromolecular aggregates to block cell uptake, and consequently have an impact on a wide array of diseases and disorders including those of nervous system, diabetes and cancer. In an embodiment, the novel compounds of the invention prevent transcellular propagation of molecules, macromolecular aggregates, protein aggregates and the like, that bind HSPGs (or similar cell surface proteins) by direct binding to a target, including but not limited to tau, alpha- synuclein, p53, IAPP, viruses and the like.

[00031] Provided herein are novel compounds related to heparinoids, wherein such novel compounds bind HSPGs. The compounds comprise pentasaccharide building blocks with “linkable linker” side chains (A) and N,N-(Disuccinimidyl) suberate (B) are used to obtain a polydisperse compound mixture of oligosaccharides (C) with a MW ranging from 2500 to 8000 Da, and containing variable numbers of the pentasaccharide building block units. The compounds may be utilized in pharmaceutical compositions for therapeutic intervention in central nervous system disorders including, but not limited to, neurodegenerative and/or prion related disorders.

[00032] As detailed in the Example section, an analog of Fondaparinux was used as a basic building block to synthesize oligosaccharides composed of multiple pentasaccharide units connected by aliphatic linkers without N-sulfation. The synthesis scheme ensured a sufficient glycosaminoglycan (GAG) chain length for tau binding (or binding of other macromolecular aggregates) and inhibition, and reduced the MW and overall charge of the resulting compounds. The inventors systematically created and tested 7 generations of compounds. The final product was SN7-13 (Figure 1), a polydisperse compound composed of 2-4 pentasaccharides with a MW from 2.5 to 8 kDa. This MW is significantly lower than the average MW of standard heparin, which ranges from 3 to 30 kDa (26,27) with considerably less sulfation than FMWH such as enoxaparin.

[00033] Consequently the inventors profiled and assessed the activity of the synthesized heparinoid, SN7-13. As noted above, SN7-13 has a low molecular weight (2.5-8 kDa), low charge, and lacks anticoagulation activity. It was created in a novel fashion based on aliphatic linkage of pentasaccharide units (Figure 2). SN7-13 has high affinity for tau, blocks tau aggregate uptake, and prevents replication in a HEK293T cells and primary neurons (Figure 12, 13). These findings suggested that a compound of this class could have therapeutic potential for certain neurodegenerative disorders, including for example, tauopathies. SN7-13 also has an affinity for other macromolecular aggregates, and as such SN7-13 and consequently has therapeutic potential for additional disorders such as cancer and diabetes.

[00034] In an embodiment, the invention comprises heparinoid-like compounds comprising oligosaccharides composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compounds have reduced sulfation. The oligosaccharides comprise 2-4 pentasaccharides wherein two or more of the pentasaccharides may be identical or non identical. The pentasaccharide units are linked in the presence of N,N-(Disuccinimidyl) suberate, the molecular weight of the compounds is 2.5 to 8 kDa, the compounds have reduced N-sulfation and diminished charge. The compounds have the ability to bind tau protein with low nanomolar affinity and have glycosaminoglycan chain length for tau binding and inhibition. Furthermore, the compounds described and claimed herein also lack anticoagulation activity.

[00035] As used herein, the term “heparinoid” comprises heparin and heparin derivatives including naturally occurring and synthetic sulphated polysaccharides of similar structure of plant, animal, or synthetic origin, including but not limited to, glycosaminoglycans. Though not wishing to be found by the following theory, it is thought that heparinoids, like heparin, act by interacting with heparin binding proteins, generally through ionic interactions or hydrogen bonding. “Fleparinoid-like” compounds generally comprise compounds and molecules that are similar to heparinoids.

[00036] In an embodiment, heparinoid-like compounds comprising oligosaccharides composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compound has reduced sulfation, are provided. The oligosaccharides may comprises 2-10, 2- 8 or 2-4 pentasaccharides wherein the pentasaccharides may be identical or non-identical. In additional embodiments, oligosaccharides may comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more pentasaccharides wherein the pentasaccharides may be identical or non identical. The pentasaccharide units may be linked in the presence of N,N-(Disuccinimidyl) suberate. The molecular weight of the compounds may be 2.5 to 8 kDa. In certain embodiments, the molecular weight of the compounds is 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 4.0, 4.5, 5.0, 5.25, 5.5, 6.0, 6.25, 6.5, 7.0, 7.25, 7.5, 8.0, 8.25, 8.5, 9.0, 9.25, 9.5, lOkDa, and any range between about 2.0 and 10 kDa, for example 2.5 to 8 kDa. In an embodiment, the reduced sulfation comprises reduced N-sulfation and in certain embodiments, the amount of sulfation is less than the amount of sulfation on heparin, and/or less than the amount of sulfation on low molecular weight heparin. In an embodiment, the charge of the compounds is less than the charge of heparin, and/or less than the charge of heparin low molecular weight. In an embodiment, the compound has the ability to bind tau protein with low nanomolar affinity, and/or the compounds have a sufficient glycosaminoglycan chain length for tau binding and inhibition. The novel compounds claimed herein lack anticoagulation activity.

[00037] In an embodiment, methods for making the novel heparinoid-like compounds described herein comprise the steps as detailed in synthesis scheme and as set forth in Figure 2, wherein the compounds comprise an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, and wherein the compounds have reduced sulfation. As contemplated herein, aliphatic linkers include, but are not limited to, alkanes, alkenes, alkynes, dienes, either saturated or unsaturated, straight or branched, and the like. In a further embodiment, methods for making heparinoid-like compounds described herein comprise the steps as detailed in the synthesis Schemes 1-6 and as set forth in Figures 1, and 4-8, wherein the compound comprises an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compounds have reduced sulfation.

[00038] In an embodiment methods for treating a subject having a central nervous system disorder, comprising administering to the subject, compositions comprising a heparinoid-like compound, wherein the heparinoid-like compound comprises an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, and wherein the heparinoid-like compound has reduced sulfation, are provided. As contemplated herein, central nervous system disorders include neurodegenerative disorders and/or prion related disorders. Central nervous system disorders may comprise a tauopathy related disorder. In addition, central nervous system disorders include but are not limited to primary age-related tauopathy (PARTj/Neurofibrillary tangle-predominant senile dementia, chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, or lipofuscinosis. In an embodiment, methods for treating a subject having a central nervous system disorder comprise administering the novel compounds described herein orally, by injection, transdermally, intravascularly (e.g., intra-arterially or intravenously), subcutaneously, intramuscularly, intra-articularly, sublingually, topically, intranasally, intracranially, by inhalation, rectally, or vaginally, among others.

[00039] Further embodiments comprise methods for inhibiting the formation of tau assemblies in a subject, comprising the administering of heparinoid-like compounds to the subject, wherein the heparinoid-like compounds comprise an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compound has reduced sulfation.

[00040] Also contemplated herein are novel pharmaceutical compositions comprising a heparinoid-like compound, wherein the heparinoid-like compound comprises an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compound has reduced sulfation. The pharmaceutical compositions described herein may be formulated into one or more dosage forms for administration orally, by injection, transdermally, intravascularly (e.g., intra-arterially or intravenously), subcutaneously, intramuscularly, intra-articularly, sublingually, topically, intranasally, intracranially, by inhalation, rectally, or vaginally, among others. The pharmaceutical compositions may comprise a liquid excipient wherein the liquid excipient may be selected from the group comprising water, DMSO, saline, buffered saline, lactated Ringer’s solution, Ringer’s acetate solution, hydroxypropylcyclodextrin solutions or ethanolic solutions. In certain embodiments, the pharmaceutical compositions may comprise a solid excipient, wherein the solid excipient is selected from the group consisting of adjuvants, antioxidants, binders, buffers, coatings, coloring agents, compression aids, diluents, disintegrants, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents, glidants, granulating agents, lubricants, metal chelators, osmo-regulators, pH adjustors, preservatives, solubilizers, sorbents, stabilizers, sweeteners, surfactants and/or bulking agents.

[00041] In an embodiment, methods for treating sickle cell disease in a subject, comprising the administering of heparinoid-like compound to the subject, wherein the heparinoid-like compound comprises an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compound has reduced sulfation, are provided. As contemplated herein, sickle cell disease is caused by sickle-shaped cells, and sickle cell disease may comprise vaso-occclusive crisis, splenic sequestration crisis, acute chest syndrome, aplastic crisis, haemolytic crisis, ischaemia, or necrosis. Symptoms associated with sickle cell disease comprise pain, anemia, swelling in extremities, bacterial infections, stroke.

[00042] In an embodiment, methods for reducing the sickling of red-blood cells in a subject, comprising the administering of heparinoid-like compound to the subject, wherein the heparinoid-like compound comprises an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compound has reduced sulfation, are provided.

[00043] In an embodiment, methods for treating and/or preventing diseases and disorders characterized by pathophysiology related to macromolecular aggregates, in a subject, comprising the administering of heparinoid-like compound to the subject, wherein the heparinoid-like compound comprises an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers, wherein the compound has reduced sulfation, are provided. As contemplated herein, diseases and disorders characterized by pathophysiology related to macromolecular aggregates may comprise cancer, diabetes, microbial, bacterial, or viral infections. Symptoms associated with such diseases and disorders comprise pain, anemia, swelling, fever, stroke, fatigue, loss of sensory perception, weight loss, weight gain, hemorrhaging, sleep disruption and the like.

Pharmaceutical Formulations

[00044] In certain embodiments, the novel compounds of described herein comprise pharmaceutical formulations. The pharmaceutical formulations comprise SN7-13 as described herein, a polydisperse compound composed of 2-4 pentasaccharides with a MW from 2.5 to 8 kDa, its enantiomers or derivatives thereof, in any amount suitable for treating neurodegenerative or prion related disorders when administered to a subject. As used herein, a “subject” is any human or animal suspected of having, suffering from or at risk for developing neurodegenerative or prion related disorders. In some embodiments, the pharmaceutical formulation may comprise SN7-13 in combination with one or more additional pharmaceutical agents: the components of the formulation may be present in varying amounts, in other embodiments, components may be present in the pharmaceutical formulations in substantially equal amounts. In other embodiments, the components are present in the pharmaceutical formulation in varying ratios of SN7-13 to a second, third etc., component: of less than 1:5, of about 1.1 to 4.5, for example about 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 and 1:4.5.

[00045] The pharmaceutical formulations described herein can be formulated into any suitable dosage form for administration to a subject. For example, the present pharmaceutical formulations can be formulated into one or more dosage forms for intravascular or intramuscular administration, for oral administration for example as a tablet or caplet, for sublingual administration or for inhalation. In one embodiment, the pharmaceutical formulations are formulated for intravenous administration.

[00046] One skilled in the art would understand how to manufacture the present pharmaceutical formulations, and how to formulate them into one or more dosage forms. For example, the present pharmaceutical formulations can be manufactured by mixing SN7-13, and optional additional components in the desired amounts, either neat or with any suitable pharmaceutical excipients. The amount and nature of the pharmaceutical excipients to be mixed with SN7-13 can be readily determined by one of ordinary skill in the art, for example by considering the solubility and other known physical characteristics of the ingredients, and the chosen route of administration. Thus, in some embodiments, the present disclosure provides methods of manufacturing a pharmaceutical formulation, or one or more dosage forms thereof, for treating neurodegenerative or prion related disorders in a subject, comprising combining SN7- 13 or its enantiomers or derivatives, with additional pharmaceutical agents. In still other embodiments, the present disclosure provides the use of SN7-13 or its enantiomers or derivatives, optionally combined with additional pharmaceutical agents to manufacture a medicament, or one or more dosage forms thereof, for the treatment of neurodegenerative or prion related disorders in a subject. As used herein, “medicament” is meant to be equivalent to “pharmaceutical formulation,” and both terms are used interchangeably.

[00047] Thus, in some embodiments, the pharmaceutical excipients used to formulate a pharmaceutical formulation or dosage form of the novel compounds described herein can be solid and/or liquid. Suitable liquid excipients are well known and may be readily selected by one of skill in the art. Such excipients can include, for example, liquid carriers such as water, DMSO, saline, buffered saline, lactated Ringer’s solution, Ringer’s acetate solution, hydroxypropylcyclodextrin solutions or ethanolic solutions. Other suitable pharmaceutical excipients which can be used to make liquid pharmaceutical formulations described herein include metal chelators, osmo-regulators, pH adjustors, preservatives, solubilizers, sorbents, stabilizers, sweeteners, surfactants, suspending agents, syrups, thickening agents and/or viscosity regulators. The liquid pharmaceutical excipients can be sterile solutions, for example when used for preparing pharmaceutical formulations for parenteral (e.g., intravenous) administration.

[00048] In some embodiments, the pharmaceutical formulations described herein can be formulated with liquid pharmaceutical excipients to form solutions, suspensions, emulsions, syrups or elixirs. In one embodiment, SN7-13 or its enantiomers or derivatives, is dissolved in a liquid carrier to form a solution. In another embodiment, for example SN7-13 or its enantiomers or derivatives, is suspended in a liquid carrier to form a suspension. In another embodiment, SN7-13 or its enantiomers or derivatives, is dissolved in the liquid carrier and additional components are suspended in the liquid carrier.

[00049] Suitable solid excipients for formulating the pharmaceutical formulations described herein are also well known to those of ordinary skill in the art. A given solid excipient can perform a variety of functions; i.e., one substance can perform the functions of two or more of the excipients described below. For example, a solid excipient can act both as a filler and a compression aid. Examples of solid excipients which can comprise the novel pharmaceutical formulations described herein include: adjuvants, antioxidants, binders, buffers, coatings, coloring agents, compression aids, diluents, disintegrants, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents, glidants, granulating agents, lubricants, metal chelators, osmo-regulators, pH adjustors, preservatives, solubilizers, sorbents, stabilizers, sweeteners, surfactants and/or bulking agents.

[00050] In one embodiment, the SN7-13 or its enantiomers or derivatives can be formulated with one or more solid pharmaceutical excipients and compacted into a unit dose form; i.e., a tablet or caplet. In another embodiment, the SN7-13 or its enantiomers or derivatives can be formulated neat or with one or more solid pharmaceutical excipients as powder or granules and added to unit dose form; i.e., a capsule. In one embodiment, SN7-13 or its enantiomers or derivatives, can be formulated neat or with one or more solid pharmaceutical excipients as a powder.

[00051] For a discussion of the properties of solid and liquid pharmaceutical excipients which are suitable for use in the present pharmaceutical formulations, see, e.g. , the excipients described in the Rowe et al., eds., Handbook of Pharmaceutical Excipients, 7th Edition, London: Pharmaceutical Press, 2012, which is incorporated herein by reference.

[00052] The solid or liquid pharmaceutical formulations described herein can be formulated or divided into one or more dosage forms for subsequent administration to a subject. For example, the one or more dosage forms can be packaged compositions; e.g., packeted powders (sachets), vials, ampoules, prefilled syringes or bags. Other suitable dosage forms include pre formed dosage forms such as tablets, caplets, capsules or suppositories. In one embodiment, the one or more dosage forms is a tablet. In another embodiment, the one or more dosage forms is a tablet comprising a pharmaceutical formulation further comprising a surfactant, a lubricant, a disintegrant, a diluent or a binder. For example, the tablet can comprise a pharmaceutical formulation and docusate sodium as a surfactant, sodium benzoate as a lubricant, sodium starch glycolate as a disintegrant, magnesium stearate as a diluent and pregelatinized starch as a binder.

[00053] The pharmaceutical formulations described herein can also be provided in dry or lyophilized forms, or as a liquid concentrate, for subsequent reconstitution or dilution into a dosage form by the addition of a suitable liquid pharmaceutical excipient. For example, a powdered, lyophilized or concentrated liquid pharmaceutical formulation can be provided in a container, to which a sterile liquid pharmaceutical excipient is added prior to (for example, immediately prior to) parenteral, e.g., intravenous, administration to a subject.

[00054] In one embodiment, the pharmaceutical compositions described herein can be utilized as inhalants. For this route of administration, the pharmaceutical compositions can be prepared as fluid unit doses comprising a vehicle suitable for delivery by an atomizing spray pump or by dry powder for insufflation. In another embodiment, the pharmaceutical compositions can be delivered as aerosols; i.e., orally or intranasally. For this route of administration, the pharmaceutical compositions can be formulated for use in a pressurized aerosol container together with a gaseous or liquefied propellant; e.g., dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and the like, for example by delivery as a metered dose in one or more actuations from a suitable delivery device.

[00055] The pharmaceutical formulations or dosage forms can be administered to a subject by any suitable route, taking into consideration factors such as the age, weight and the overall condition of the subject, the type of disorder to be treated, and the like. For example, the pharmaceutical formulations or dosage forms can be delivered orally, by injection, transdermally, intravascularly (e.g., intra-arterially or intravenously), subcutaneously, intramuscularly, intra-articularly, sublingually, topically, intranasally, by inhalation, rectally, and vaginally, among others.

[00056] The pharmaceutical formulations or dosage forms can contain any amount of SN7- 13 or its enantiomers or derivatives suitable for treating neurodegenerative or prion related disorders. In one embodiment, the pharmaceutical formulations or dosage forms described herein can comprise about 10 to 1600 mg of SN7-13, for example about 10, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500 or 1600 mg of SN7-13. In one embodiment, the pharmaceutical formulations or dosage described herein can comprise about 10 to 320 mg of SN7-13, for example about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 220, 300 or 320 mg. In other embodiments, the amount of SN7-13 or its enantiomers or derivatives, comprising the pharmaceutical formulations or dosage forms is calculated as about 1/2 to 1/4, for example about 1/2, 1/3 or 1/4 the amount of other components present.

[00057] The pharmaceutical formulations and dosage forms can be administered to a subject to treat neurodegenerative or prion related disorders such as tauopathies, characterized by neuronal and/or glial inclusions composed of the microtubule-binding protein, tau. Such disorders include, but are not limited to, primary age-related tauopathy (PART)/Neurofibrillary tangle-predominant senile dementia, (with NFTs similar to AD, but without plaques,) chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked to chromosome 17, Lytico-bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[00058] Described herein is a method of treating neurodegenerative or prion related disorders in a subject, comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as provided, or one or more dosage forms thereof.

[00059] As used herein, a “therapeutically effective amount” of a pharmaceutical formulation, or one or more dosage forms thereof, is any amount which treats the neurodegenerative or prion related disorder. As used herein, to “treat” a neurodegenerative or prion related disorder means that neurodegenerative or prion related disorder is observed, and/or that one or more symptoms of the neurodegenerative disorder or prion related (e.g., memory, disorientation, confusion, muscle control) are reduced, ameliorated or delayed. [00060] The ordinarily skilled physician can readily determine the therapeutically effective amounts of the a pharmaceutical formulation or dosage form for administration to a subject according to the present methods, for example by taking into account factors such as the specific neurodegenerative or prion related disorder to be treated, and the size, age, weight, gender, progression of the disorder, route of administration, previous treatments and response pattern of the subject.

[00061] Suitable dosages and ratios of the pharmaceutical compositions may be determined based on the relative pharmacokinetics and plasma half-life (tl/2) of SN7-13 and other components.

[00062] A therapeutically effective amount of the pharmaceutical formulations or dosage forms can be administered to a subject on regular schedule; i.e., a daily, weekly or monthly at regular intervals, or on an irregular schedule with varying administration over days, weeks, or months. Alternatively, the therapeutically effective amount of the present pharmaceutical formulations or dosage forms administered can vary between administrations. For example in one embodiment, the amount for the first administration is higher than the amount for one or more of the subsequent administrations. In another embodiment, the amount for the first administration is lower than the amount for one or more of the subsequent administrations. [00063] A therapeutically effective amount of the pharmaceutical formulations or dosage forms can be administered over various time periods, for example about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every two weeks, about every three weeks, about every month, and about every two months. The number and frequency of dosages corresponding to a course of therapy can be determined according to the judgment of the ordinarily-skilled physician. The therapeutically effective amounts described herein can refer to a single administration of the pharmaceutical formulations or dosage forms, or can refer to the total amounts administered for a given time period.

[00064] In some embodiments, effective amounts of the pharmaceutical formulations or dosage forms are administered to a subject, for example as equally divided doses or as unequally divided doses, about every 24 hours for 5 days; every 12 hours for 3 days; every 12 hours for 5 days; every 6 hours for 10 to 21 days; every 12 hours for 14 days, every 12 hours for 10 days; 3 or 4 equally divided doses every 6 to 8 hours for up to 14 days; 2 or 4 equally divided doses every 6, 8 or 12 hours for up to 14 days. In another embodiment, effective amounts of the pharmaceutical formulations or dosage forms are administered to a subject, for example as equally divided doses or as unequally divided doses, for about 5 to 14 days, from one to three times a day.

[00065] Also provided herein are kits comprising one or more dosage forms comprising pharmaceutical formulations. The kits can optionally comprise instructions to direct a health care professional or a subject to prepare, store and/or administer the one or more dosage forms. [00066] Suitably, the kit contains packaging or a container with the one or more dosage forms formulated for the desired route of administration. Other suitable components comprising kits will be readily apparent to one of skill in the art, taking into consideration the desired indication, type of dosage form and the desired delivery route. A number of packages or containers are known in the art for dispensing the one or more dosage forms. In one embodiment, the package comprises indicators to assist in monitoring the delivery schedule for the one or more dosage forms. In another embodiment, the package comprises a blister package, dial dispenser package, bottle, vial, ampoule or flexible bag.

[00067] The packaging comprising a kit can itself be engineered to perform or assist in the administration of the one or more dosage forms, and can comprise for example a catheter, syringe, pipette, flexible IV bag optionally with tubing, metered dosing device for inhalation or insufflation or other apparatus from which the one or more dosage forms can be applied to or into the subject, or to or into an affected area of the subject. [00068] In some embodiments, the one or more dosage forms comprising kits are provided in dried, lyophilized or concentrated forms. In such embodiments, the kits can further comprise reagents or components for reconstitution or dilution of the one or more dosage forms. In other embodiments, the kits can comprise a means for containing the one or more dosage forms in close confinement for, e.g., commercial sale, such as injection or blow-molded plastic containers into which the one or more dosage forms are retained.

EXAMPLES

[00069] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are illustrative only, since alternative methods can be utilized to obtain similar results.

Example 1

Synthesis and Assessment of Novel Heparinoid Like Compound, SN7-13 [00070] Heparin has commonly been used as a proxy analogue of Heparan sulfate (HS). The potency of heparin to interfere with HS - HS active proteins (HSAPs) has been exploited to probe the role of heparan sulfate in biological processes. The heterogeneous nature of the sulfated oligosaccharides that heparin is made of, and its interactions with a slew of the biologically important ligands, including antithrombin III, has made it less favorable to be used in drug discovery. The sulfated pentasaccharide fondaparinux is one of the earliest and a short oligosaccharide that has specifically been recognized by a single active protein, antithrombin III (8, 9, 43). The unique characteristics of iduronic acid, glucuronic acid, rare tri-sulfated glucosamine, all present in this short pentasaccharide makes this conformationally flexible heparinoid attractive to be explored in probing the biological activity of other HASPs (47). One important structural feature of the fondaparinux molecule in altering the coagulation cascade is the presence of three N-sulfate moieties. Stripping the molecule from N-sulfate groups has been shown to drastically minimizes its anticoagulation characteristics (24, 25). The objective in producing heparin-like oligosaccharide molecules based on monomeric non-N-sulfated fondaparinux (Figure 1, scheme 1, compound 1; commonly named N-l Fondaparinux) is to generate polymeric “pentasaccharide” oligosaccharide chains with a defined and flexible heparin-like sequence. In this work, synthesis and characterization of such “pentasaccharide” derived heparinoids are described. Materials and Methods

Protein Preparation, Fibrillization And Labeling

[00071] Recombinant full-length wild-type tau was purified and fibrillized as described previously (1). For uptake assays, fibrils were incubated with Alexa Fluor 647 succinimidyl ester dye (Invitrogen) for 1 hr at RT (molar ratio monomer/dye 1:12.5), quenched with 100 mM glycine for 1 hr at RT and dialyzed overnight into PBS using dialysis cassettes (Thermo Scientific) to remove excess dye. The labeled fibrils were stored at 4°C until use.

Synthesis And Quality Control Of SN7-13

[00072] The synthesis (Figure 1) was performed at PharmaRen in St. Louis, MO. A 200 mg of a building block pentasaccharide and 400 mg NaFIC0 ₃ were dissolved in 8 ml dlFLO. Separately, 252 mg of N,N-(Disuccinimidyl) suberate was dissolved in 7 ml DMF. 4 ml of this solution was added to the aqueous solution of the pentasaccharide derivative in 4 portions in 8 hrs. The mixture was allowed to stir at RT overnight. The next day, the remaining 3 ml DMF solution was added in two portions and the mixture was stirred for 4 hrs. The reaction mixture was evaporated and extracted with EtOAc (3x15 ml). The aqueous layer was evaporated to dryness and re-dissolved in 10 ml dlFLO and purified on Sephadex G 25 column (Sigma Aldrich). The fractions that were stained blue by treatment with ammonium molybdate stain (TCI Chemicals) on a silica gel thin layer plate were combined and evaporated to produce 140 mg of glassy off- white flakes, yield: 140 mg. The reproducibility of the described procedure was confirmed by repeating the experiment following the protocol precisely at different scales. The compound was subsequently dissolved at 10 mg/ml in PBS and stored in aliquots at 4°C until further use. The synthesis of the pentasaccharide building block, as well as extensive quality control data is detailed herein. SN7-13 is a polydisperse compound and the molecule weight ranges from 2.5 to 8 kDa (Figure 2 and 3). For the calculations of concentrations in our cell-based assays and the analysis of the kinetic studies in this paper, we assumed an average MW of 5 kDa.

Di-(pentasaccharide) Compound (2)

[00073] The presence of three free amino groups in N-l Fondaparinux (Compound 1) offered the opportunity for the synthesis of novel synthetic HS analogues. In pursuing this goal, N-l Fondaparinux (Compound 1), was treated with N, N (Disuccinimidyl) suberate (reagent I in Scheme 1 (Figure 1)) and the major product proved to be a dimeric pentasaccharide in 75% yield. The structure of this Di-(pentasaccharide) was extensively studied by 2-D NMR and unambiguously proved to be a linear (in a conventional 1 4, head-to-tail direction) di- (pentasaccharide) heparinoid as depicted in scheme 1. Encouraged by this finding, several other experiments were designed and carried out to examine if by changing the experimental conditions, higher orders of oligomerization of Compound (1), and therefor longer and higher MW heparinoids, can be attained. Although mass spectrometry analyses of the products from several repeated preparations confirmed the formation of some tri- and tetra-pentasaccharide oligomers, these were only found in minute amounts and most often with poor reproducibility. The main product proved to be di-(pentasaccharide). Flowever, the results from tau uptake assays demonstrated that the di-(pentasaccharide) molecule and many of its derivatives (structures are not shown) were frequently failing to produce notable biological activities and only occasionally and in very few samples significant activity was observed. Since multiple rounds of purification of di-(pentasaccharide) did not show any improvement in the biological activities of compound (1), we reasoned that occasional observations of the biological activities in some samples may be due to presence of the contaminations from higher oligomers of the Pentasaccharide (e.g. tri- and tetra-(pentasaccharide) which were forming at random during some oligomerization reactions. Several unfruitful attempts in producing higher order oligomers in a consistent and reproducible fashion from building block (1), led us to design a more active pentasaccharide building block (as described in the next paragraph below). Interestingly, biological activity was not observed when preparing compound (3) through N- sulfation of compound (2). Thus, the addition of sulfate moieties does not necessarily increase the biological potency of a compound.

Preparation Of Oligomeric Pentasaccharides

[00074] Though not wishing to be bound by the following theory, it was reasoned that the isolation of mainly a single linear di-(pentasaccharide) molecule (in a 1 4 like saccharides chain) from several theoretically possible derivatives is most likely the result of: a) the variation in the basicity/reactivity of the three amino groups in Compound (1) and; b) possible electrostatic negative charge repulsive interactions of the sulfate groups that result in forcing the molecule to adopt a linear (versus branched) configuration. To increase the basicity of the amino groups at the C-2 position of the glucosamine sugars, an amino linker was appended to each amino group through an amide bond via the reaction with 6-[N-(tert-butoxycarbonyl]-l- (N-hydroxysuccinimidyl) hexanoate (reagent II in scheme 6 (Figure 8)). By subsequent deprotection of the product, the inventors generated the new pentasaccharide building block, a molecule with three primary aliphatic amino chains and with almost equivalent basicity (compound 4, scheme 3). The presence of three amino linkers in this molecule was further confirmed by its conversion to the corresponding N-sulfated derivative (compound 6, Scheme 5 (Figure 7)) which was fully characterized by 2-D NMR and Mass spectrometry. The new building block (compound 4) on reaction with di-(N-succinimidyl) suberate (reagent I), and followed by subsequent purification, produced products (compound 5, scheme 4 (Figure 6)) which on analyses by size exclusion chromatography and mass spectrometry proved to be a mixture oligosaccharides of higher orders. To demonstrate the reproducibility of the protocol, several preparations of the new heparinoid were performed and the products of each preparation (Batches 1-5, scheme 4) exhibited high consistency in terms of the molecular size and mass distribution (Figure 9). They all revealed high potency in their biological activities (tau uptake inhibition similar to heparin) and importantly displayed minimal anticoagulation activity. The characterization of the new heparinoids (6-9) was performed with a combination of chromatography and mass spectrometry techniques. Strong Anion Exchange chromatography followed by Size Exclusion chromatography proved that the synthetic oligosaccharides have higher MW than the di-pentasaccharide molecules (2 and 3) . The presence of the linkers made the direct comparison of SEC data of the new heparinoids with that of the known LMWFI reference Enoxaparin (with an average MW of 4500 Da) less straight forward. Flowever, mass spectrometry data of the oligosaccharides (5) established that MW is in the range of 2500-8000 and composed of a number of oligosaccharides with variable length including tri- and tetra-pentasaccharide derivatives.

Experimental: Preparation of Compounds (l)-(5)

[00075] Preparation of Compound (1)- Fully protected pentasaccharide was prepared according to the published procedure and was subjected to the sequential reactions of hydrolysis, O-sulfonation, and hydrogenation as previously published (21, 22). The crude reduced and sulfated product was purified on a SAX column and desalted on a size exclusion column.

[00076] Preparation of Compound (2), scheme 1- Compound 1, 200 mg, 0.14 mmol, 1 equivalent was dissolved in 5 mL dIFI20. N, N-(disuccinimidyl) suberate (TCI Chemicals), 26 mg, 0.07 mmol, 0.5 equivalent was dissolved in 1 mL DMF and added to the aqueous solution of the pentasaccharide. To the solution was added 160 mg, 1.9 mmol, 13.6 equivalent of NaHC03. The mixture was stirred at room temperature overnight. Reaction mixture was evaporated to dryness and re-dissolved in 10 mL dlFLO and subjected to SAX column (16 g. BioRad Macro-Prep HiQ resin in 100 mL dlFLO) and eluted with 0-2M gradient sodium chloride. Fractions that were stained blue on silica gel plates on treatment with ammonium molybdate stain solution were combined and evaporated to dryness. The salty residue was dissolved in 10 mL dlFLO and desalted on Sephadex G-25 (Sigma- Aldrich, 200 mL in a 100 cm column). The desalted fraction that stained blue on treatment with the molybdate stain solution were combined and evaporated to a glassy colorless flakes. The product was one more time purified as described above to obtain a product that reached a purity >75%. Yield: 52 mg. [00077] N- Sulfation of Compound (2)-Preparation of compound (3), Scheme 2- Compound (2), 100 mg, 0.07 mmol, 1 equiv., was dissolved in 5 mL dlFLO. To the aqueous solution was added Na2C(¾ to adjust the plT to about 10. Sulfur trioxide:Trimethylamine complex, 200 mg, 1.4 mmol, 20 equiv., was added in portions to the aqueous solution at 50°C, and by additional addition of 1M Na ₂ C0 ₃ , kept the plT at 9- 10. After completion of the addition of sulfur trioxide (about 2-3 hrs), the reaction mixture allowed to cool to room temperature and stirred overnight. The reaction mixture was extracted with EtOAc, 20 mL, and the aqueous solution was fractionated on a SAX column and de-salted on G-25 column as described earlier. The product, 140 mg, was evaporated to a slightly pink, and glassy flakes.

[00078] Preparation of the Reagent (II)- (Scheme 6). 6-Aminohexanoic acid (650 mg, 5 mmol, 1 equiv.) was placed in an RB flask and 5 mL 1T20 was added. To the aq. solution at room temperature was added 4 mL T1TF and 1.5 mL Et3N. Lightly turbid solution was observed. To the solution was added 1.3 mL t-Boc Anhydride (1.1 equiv.). The reaction mixture was vigorously stirred at room temperature overnight.

[00079] The clear colorless solution of the reaction mixture was evaporated. Acidified to plT 3 by addition of 2N FICl and extracted with 15 mL EtOAc (2X). EtOAc layer was dried over MgSOt, filtered and dried on rotavapor. NMR studies confirmed the product structure.

[00080] The residue was dissolved in 15 mL DCM and subjected to the next reaction. In 10 mL DMF solution, HOBt (7 mmol, 1.4 equiv., 946 mg), EDICDI (1.4 equiv., 7 mmol, 1.35g) was dissolved. DCM solution of the t-Boc protected 6-Aminohexanoic acid was added to the DMF solution and the mixture was stirred at room temperature for 2 hrs. To the solution was added N-hydroxy succinimide (1.6 equiv., 8 mmol, 920 mg) and the mixture was stirred at room temperature for 18 hrs. The mixture was evaporated to dryness. The residue (colorless thick liquid) was dissolved in 25 mL Acetonitrile. After an hour, white crystals precipitated. The ACN solution was decanted and a sample was examined by HPLC (Supplementary Materials). Acetonitrile solution was evaporated and re-dissolved in 10 mL DCM. Although the product does not stain well and is not detectable immediately by UV light, however, after exposing to UV light for 10-18 hrs, an intense spot under UV light can be observed. Silica-gel chromatography (40-100% Hex-EtOAc) afforded the desirable product (Rf=0.6, 60% H-E). Yield: lg.

[00081] Preparation of Compound (4), Scheme 3. Compound (1), N-l Fondaparinux, 500 mg, and 900 mg of NaHC03 were dissolved in 15 mL of dIH20. To this clear solution was added a solution of 700 mg of Reagent (II) in 7 mL DMF. The cloudy heterogeneous solution was momentarily warmed up in a water bath and then stirred at room temperature. The solution gradually became a clear solution. After 6 hrs, to the clear solution was added another 700 mg of the reagent (I) to the reaction mixture and the heterogeneous solution was stirred at room temperature overnight. Reaction mixture was evaporated to dryness and extracted with 25 mL dIH20 and 3X10 mL EtOAc. The aqueous layer was separated, and the pH was adjusted to 7. After one more additional extraction with 20 mL EtOAc, the aqueous layer was treated with 2.5 mL TFA at room temperature for 3 hrs. The mixture was fractionated on a Hi Q column and the fractions that were stained blue with Molybdate stain were combined and desalted oh G-25 column. The 320 mg of the product was obtained with a purity of 78% as confirmed by HPLC data.

[00082] Compound (4) was fully N-sulfated as described for the compound (3) and the product, compound (6, scheme 5) and the purified material was characterized by mass spectrometry and 2-D NMR.

[00083] Preparation of Compound (5). Compound (4), 200 mg, 114 pmol, 1 equiv. and NaHC03, 400 mg, 42 equiv. were dissolved in 8 mL dIH20. Separately, N, N- (Disuccinimidyl) suberate, 252 mg, 0.68 mmol, 6 equiv., was dissolved in 7 mL DMF. 4 mL of this solution was added to the aqueous solution of the pentasaccharide derivative in 4 portions in 8 hrs. The mixture allowed to stir at room temperature overnight. The next day, the remaining 3 mL DMF solution was added in two portions and the mixture was stirred for 4 hrs. The mixture was evaporated and extracted with EtOAc (3x15 mL). The aqueous layer was evaporated to dryness and re-dissolved in 10 ml dl¾0 and purified on G 25 column. The fractions that were stained blue on treatment with ammonium molybdate stain on a silica gel thin layer plate were combined and evaporated to afford 140 mg of glassy off- white flakes, yield: 140 mg. The reproducibility of the procedure described here was confirmed by repeating the experiment following the protocol precisely as described here at different scales and the results are collected in the following table.

Example 2

Assessment of Novel Heparinoid Like Compounds Coagulation studies: In vitro spiking of normal pooled plasma

[00084] Coagulation studies were carried out at the clinical laboratory of Children’s Medical Center Dallas, Texas. To assess the anticoagulative properties of SN7-13, an in vitro spiking experiment was performed. Normal pooled plasma was used for spiking with increasing concentrations of SN7-13 and porcine unfractionated heparin (AMSBio). Normal pooled plasma was aliquoted into 1 ml microtubes. Increasing concentrations (0.01 ug/ml, 0.1 ug/ml, 1 ug/ml, 10 ug/ml, 20 ug/ml, 50 ug/ml, 100 ug/ml) of SN7-13 and unfractionated heparin were added to the normal pooled plasma. The activated partial thromboplastin time (PTT) and prothrombin time (PT) were analyzed within 30 min. The PTT and PT assays were performed on the STA-R Evolution (Diagnostica Stago, New Jersey). The PT reagent used was STA Neoplastine Cl Plus and the PTT reagent was STA-PTT Automate reagent. An abnormal PTT was defined as a result outside the laboratory’s normal range (21.3-38.8 seconds). An abnormal PT was defined as outside the range of 12.0 to 15.3 seconds.

Bio-Layer Interferometry

[00085] Bio-layer interferometry was used to characterize the interaction of tau monomer with SN7-13 versus heparin as a control. Assay buffer was composed of 10 mM HEPES pH 7.4, 100 mM NaCl, 0.1% BSA and 0.01% Tween 20 in deionized water. Full length wild-type tau monomer was prepared as referenced above ( 1 ) and biotinylated with EZ-Link NHS-PEG4- Biotin (Thermo Scientific) according to the manufacturer’s instructions. For 500 mΐ of 1.656 mg/ml tau monomer, the amount of 10 mM biotin solution was calculated using the formula (MCR = Molecular Coupling Ratio):

1 656771 Q / 771 L ( C071C uL of 10 mM biotin Reagent = — ( MCR ) x 500 uL ( vol ) /10 = 9 uL

[00086] Tau monomer was incubated with the required amount of biotin for 60 min at RT. The reaction was quenched using ZEBA spin columns 7K MWCO (Thermo Scientific) according to the manufacturer’s instructions, and subsequently stored at -80 °C in small aliquots until use. For experiments, biotinylated tau monomer was diluted with assay buffer to a concentration of 0.2 uM. In addition, biocytin (Sigma) was diluted in assay buffer to a stock solution of 1 mM. Super Streptavidin biosensors (ForteBio) were equilibrated in 200 mΐ of assay buffer 20 min before using in a 96-well plate (Greiner). Meanwhile, 50 mΐ of assay buffer, biotinylated tau fibrils, biocytin, and serial dilutions of heparin (innM: 10, 5, 2.5, 1.25, 0.625) and SN7-13 (in nM: 200, 100, 50, 25, 13) were transferred to the designated wells on the 384- well assay plate, and the plate was centrifuged for 1 min at 1000 rpm. Binding assays were performed by loading the sensors with biotinylated tau monomer, followed by quenching of streptavidin on the sensor with biocytin and equilibration in assay buffer, an association step (300 s) and a dissociation step (600 s) using an Octet RED 384 system (ForteBio, Pall Fife Sciences) and the Octet 8.2 Data Acquisition software. Assays were performed at 30°C and 1000 rpm shaking. For data processing, data from reference sensors (not loaded with tau) was subtracted from sensors loaded with tau to account for any non-specific binding of heparin or SN7- 13 to the sensors. In all experiments performed here, heparin and SN7- 13 showed no non specific binding to unloaded Super Streptavidin sensors. Processed data were fit globally (across all dilutions within an experiment) to a 1:1 binding model to obtain kinetic and thermodynamic parameters. Data were processed and analyzed in the Octet Data Analysis 8.2 software and graphs were plotted in GraphPad Prism v8 for Mac OS X.

Cell culture of HEK293T cells and P301S FRET biosensor cell line

[00087] The inventors previously used HEK293T cells to generate a stable monoclonal P301S FRET biosensor cell line by overexpressing tau repeat domain (RD) with the disease- associated mutation P301 S, and tagged at the C-terminus with either CFP or YFP to detect tau seeding (ATCC CRF-3275) (30,31). All HEK293T cells were grown in complete media: Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) with 10% fetal bovine serum (Sigma), 1% penicillin/streptomycin (Gibco) and 1% Glutamax (Gibco). Cells were cultured and passaged at 37°C, 5% CO2, in a humidified incubator. Dulbecco’s phosphate buffered saline (Life Technologies) was used for washing the cells prior to harvesting with 0.05% Trypsin-EDTA (Life Technologies).

Tau Uptake Assay

[00088] Uptake assays were performed as described previously (2). HEK293T cells were plated at 20,000 cells per well in a 96-well plate in 100 mΐ of media per well. Fluorescently labeled aggregates (100 nM monomer equivalent) were sonicated for 30 s at an amplitude of 65 (corresponding to ~80 Watt; QSonica) and pre-incubated overnight at 4°C in media containing standard heparin (AMSBio) or SN7-13 at 5 different concentrations (0.2 - 500 pg/ml). The following morning, the aggregate-GAG complexes were applied to cells for 4 hrs in media vehicles of 50 mΐ per well. Cells were harvested with 0.25% trypsin for 5 min, fixed with 2% PFA for 10 min, and resuspended in flow cytometry buffer (HBSS plus 1% FBS and 1 mM EDTA) before flow cytometry. Cells were counted with the LSRFortessa SORP (BD biosciences). We determined the median fluorescence intensity (MFI) per cell to quantify cellular aggregate internalization. Each experiment was conducted in three independent biological replicates, with technical triplicates per condition. We determined the average mean fluorescence intensity (MFI) of the replicates for each condition and standardized to aggregate uptake without inhibitor treatment within each experiment. The standardized averages of each condition were then combined. Data analysis was performed using FlowJo vlO software (Treestar Inc.) and GraphPad Prism v8 for Mac OS X.

Tau Seeding Assay

[00089] The inventors used the P301S FRET biosensor cell line for seeding experiments (30,31). The seeding assay was conducted as described except that biosensor cells were plated at a density of 15,000 cells/well in a 96-well plate in a media volume of 100 ul per well. Recombinant tau fibrils were sonicated for 30s at an amplitude of 65 (corresponding to ~80 Watt, QSonica) prior to use. Fibrils (100 nM monomer equivalent) were pre-incubated overnight at 4°C in media containing the standard heparin (AMSBio) or SN7-13 at 5 different concentrations (0.2 - 500 pg/ml). At 20-30% confluency, the seed-heparin complexes were applied to the cells in media volumes of 50 pi per well, and cells were incubated for an additional 84 hrs. Fipofectamine to was not used to drive internalization of seeds since HSPG- mediated uptake was to be monitored. Cells were harvested with 0.05% trypsin and fixed in 2% PFA for 10 min, then resuspended in flow cytometry buffer (FIBSS plus 1% FBS and 1 mM EDTA). The LSRFortessa SORP (BD Biosciences) was used to perform FRET flow cytometry. FRET was quantified as previously described (30,31) with the following modification: single cells were identified that were YFP- and CFP-positive and subsequently quantified FRET positive cells within this population. For each data set, 3 independent experiments with 3 technical replicates were performed. Data analysis was performed using FlowJo vlO software (Treestar Inc.) and GraphPad Prism v8 for Mac OS X.

Primary Neuron Culture

[00090] The day before preparation of the neurons, plates were coated with 0.05% polyethyleneimine (PEI) in 25 mM borate buffer, pH 8.4 at 37°C overnight and washed 3 times with PBS the next day. Hippocampal neurons were isolated from El 7.5 CD1 mice as previously described (48). The neurons were incubated with 0.5% trypsin-EDTA (Gibco) in HBSS (Gibco) with 1 :6 glucose (Sigma) for 20 min at 37°C. Neurons were then triturated and filtered through a 70 pm filter (Greiner Bio-one - Easy strainer), followed by plating 30,000 cells per well of a 96-well plate in plating media: MEM (Gibco), 10% FBS (Gibco), 1 mM pyruvate (Gibco), 37% glucose (Gibco), 1% penicillin-streptomycin (Gibco). After a 3 hr incubation period, the media was completely exchanged by 3-hr exchange media: Neurobasal Medium (Gibco), B27 (Gibco), 200 mM GlutaMAX (Gibco), 25 mM Glutamic Acid (Sigma). After 3 days and from there on twice weekly, 50% of the media in each well was replaced with 3-day exchange media: Neurobasal Medium (Gibco), B27 (Gibco), 200 mM GlutaMAX (Gibco).

Tau Uptake Assay In Primary Neurons

[00091] Tau uptake assays in primary hippocampal neurons were conducted similarly to the uptake assay in HEK293T cells described above, with the following modifications: Neurons were incubated with tau fibrils for 16 hrs overnight, and the tau/compound complexes were applied in vehicle at 100 pi per well (total volume per well was 200 pi).

Results

Synthesis Of A Heparin Derivative With Low Molecular Weight

[00092] After prior work determined a lower limit of GAG length that binds tau (2), the inventors decided to use a pentasaccharide analog of fondaparinux, a low molecular weight anticoagulant, as the starting molecule (scheme 1 (Figure 1). The inventors had previously established protocols to allow efficient and scalable synthesis of this compound (21,22). [00093] Fondaparinux binds antithrombin (8,9,23). Three N-sulfate moieties play a critical role, as their deletion significantly reduces its anticoagulation properties (24,25). It was previously found that N-sulfation of GAGs mediates binding to tau, and is required for its cellular uptake (7). Surprisingly, the inventors determined that replacing N-sulfate moieties with “linkable linkers” retained tau binding and inhibitory activities similar to heparin (Figure 1). The inhibitory potency of heparin derivatives on tau uptake increases with sugar chain length had been previously determined (25). Thus, several pentasaccharides were linked by carbon chains (Figure 1), extending the GAG length without increasing the overall charge of the molecule.

[00094] Ultimately, an analog of fondaparinux was used as a basic building block to synthesize an oligosaccharide composed of multiple pentasaccharide units connected by aliphatic linkers without N-sulfation. This ensured a sufficient GAG chain length for tau binding and inhibition, and reduced the MW and overall charge. The inventors systematically created and tested 7 generations of compounds. The final product was SN7-13 (Figure 1), a polydisperse compound composed of 2-4 pentasaccharides with a MW from 2.5 to 8 kDa. This MW is significantly lower than the average MW of standard heparin, which ranges from 3 to 30 kDa (26,27) with considerably less sulfation than LMWH such as enoxaparin.

SN7-13 Does Not Affect The Coagulation Cascade

[00095] The inventors first determined the coagulation properties of SN7-13 in comparison to heparin (Table 1). Like heparin, SN7-13 did not affect the prothrombin time (PT). However, while heparin dose-dependently increased the partial thromboplastin time (PTT) at therapeutic concentrations, SN7-13 had no activity, even at high concentrations (100 pg/ml).

Table 1

Table 1: SN7-13 does not affect the activated PTT or PT. The PTT and PT were determined in a routine laboratory for human diagnostics using normal pooled plasma spiked with heparin and SN7-13 at the indicated concentrations (Children’s Medical Center Dallas). Both PTT and PT were measured in seconds. The normal range of the PTT was defined as 21.3-38.8 seconds, and of the PT as 12.0 to 15.3 seconds. Abnormal results of the PTT are shaded in red for heparin. The corresponding PTT results for SN7-13 at the same concentrations fall in the normal range and are shaded in green. Heparin and SN7-13 do not affect the PT.

SN7-13 Binds Tau With Low Nanomolar Affinity

[00096] A bio-layer inferometry (BLI) kinetic binding assay (ForteBio) was used to analyze binding of tau monomer to SN7-13 in comparison to heparin (Figure 3 and Table 2 (below)). The tau monomer was biotinylated and immobilized on Super Streptavidin biosensors, which were subsequently exposed to serial dilutions of heparin or SN7-13. Analysis of the binding kinetics indicated that heparin bound tau with very high affinity, with an apparent KD = 60 pM +/- 30 pM (Figure 3A). The interaction had a fast on-rate (~ 9 x 10 ⁵ M V ¹ ) and slow off-rate (~ 6 x 10 ⁵ s ¹ ). In comparison, SN7-13 bound to tau with an apparent KD = 2 nM +/- 1 nM (Figure 3B). The interaction had a fast on-rate (~ 1.6 x 10 ⁵ M ^_1 s ^_1 and slow off-rate ~ 3.1 x 10 ⁴ M V ¹ ), qualitatively similar to heparin. In summary, SN7-13 demonstrated high binding efficiency for tau, albeit slightly weaker than heparin.

Table 2

Table 2: Results of bio-layer inferometry kinetic assay for binding of tau to heparin and SN7-13.

Both heparin and SN7-13 bind to tau with high affinity with a K _D in the picomolar (heparin) and nanomolar (SN7-13) range. The results were calculated as the mean of 4 independent experiments +/- SD.

SN7-13 blocks tau uptake in HEK293T cells

[00097] The inventors had previously observed that heparin prevents the binding of tau to HSPGs on the cell surface, blocking aggregate uptake via HSPG-mediated macropinocytosis (1,2). To test the efficacy of SN7-13, the inventors labeled recombinant tau fibrils with Alexa 647 dye and exposed HEK293T cells in culture to the fibrils for 4 hrs, titrating concentrations of heparin and SN7-13 (Figure 12A). Aggregate uptake was quantified by measuring the median fluorescence intensity (MFI) per cell using flow cytometry (1). Heparin reduced tau uptake by -50% even at the lowest concentration tested (0.2 pg/ml). Higher concentrations of heparin decreased tau uptake virtually to zero. SN7- 13 had slightly lower potency, but inhibited uptake similarly to heparin.

SN7-13 blocks tau uptake in mouse hippocampal neurons

[00098] SN7-13 was tested to determine if it would inhibit tau uptake in primary cultured neurons. Mouse hippocampal neurons from El 7.5 CD1 mice were isolated and cultured in 96- well plates at 30,000 neurons per well. Between DIV 7 and 14, they were exposed to labeled tau fibrils for 16 hrs, and quantified tau uptake by measuring the MFI via flow cytometry. Heparin and SN7-13 both dose-dependently reduced tau uptake (Figure 12B).

SN7-13 Blocks Seeding With Recombinant Tau

[00099] Multiple pathways for cellular tau uptake and downstream pathways have been proposed (28,29), and some may lead to the generation of seeding in the cells, while others may not. Therefore, SN7-13 was tested to determine if it blocks tau seeding in a well- characterized “biosensor” cell line (Figure 13A). These cells stably express the tau repeat domain (RD) containing the disease-associated mutation P301S, and tagged with CFP or YFP (30,31). When exposed to extracellular tau fibrils, tau aggregates bind to the cell surface, trigger their own uptake, and induce intracellular aggregation of tau RD-CFP/YFP, bringing the fluorophores in close proximity and enabling fluorescence resonance energy transfer (FRET). FRET was used to measure intracellular seeding by flow cytometry. The inventors exposed tau fibrils to heparin or SN7-13 for 2 hrs prior to addition to the biosensor cells. These were incubated for 72 hrs, and harvested for flow cytometry. Both heparin and SN7-13 dose dependently reduced seeding, with similar potency (Figure 13 A).

SN7-13 Blocks Seeding By P301S Transgenic Mouse Brain Homogenate [000100] The inventors had previously studied the progressive tau seeding activity that develops in a transgenic mouse line based on expression driven by the prion promoter of 1N4R human tau containing the disease-associated mutation (P301S), termed PS 19 (31-33). Brain homogenates from ~ 400 to 450 day-old animals were created and applied it to the biosensor cells (33). Lysate was incubated with heparin or SN7-13 in increasing concentrations for 2 hrs prior to addition to the cells. After 72 hrs intracellular aggregation was quantified by FRET flow cytometry (31). Fleparin dose-dependently reduced seeding by brain homogenates. SN7- 13 reduced seeding similarly to heparin at higher concentrations (Figure 13B).

Discussion

[000101] The inventors have previously proposed that transcellular propagation of tau aggregates underlies progression of neurodegeneration in Alzheimer’s Disease and related tauopathies (34). They have further determined that tau binding to FISPGs at the cell surface mediates uptake and subsequent pathological conversion of native protein on the cell interior (1,2). The inventors have now developed a unique inhibitor of tau uptake, SN7-13, that targets this mechanism. While heparin and LMWH have been used in humans as potent anticoagulants for many decades (26, 27), the therapeutic window for both is relatively narrow, overdose can result in hemorrhage (6), and neither is a plausible agent to inhibit transcellular propagation of tau pathology.

[000102] Fleparin is polydisperse with MW of 3-30 kDa (average 15 kDa). Its size and charge would be expected to prevent it from passing the blood brain barrier (6). LMWH has a MW of 1-lOkDa (average 4.5 kDa) (6), and preparations of heparin-derived oligosaccharides can pass the blood brain barrier with CSF to plasma ratios of ~1% (35,36). LMWH has a longer half- life, better bioavailability at low doses, and a more predictable dose-response than heparin (6). [000103] Heparin and heparin derivatives have been previously studied for their potential to treat neurodegenerative disorders associated with prion protein, tau and Abeta. Early observations reported that LMWH and polysulfated compounds inhibit Abeta binding to heparin and HSPGs (36,37), and that heparin inhibits the binding of APP to HSPGs (38) in vitro. The LMWH neuroparin has exhibited neuroprotective effects in several animal models of Alzheimer’s Disease (AD) (18), and similar effects were observed for enoxaparin (19,20). For prion diseases, studies in the 80s and 90s reported that sulfated glycans inhibit the cellular uptake of prion protein aggregates in cell culture, and were proposed as potential treatments (7,12,13,39). Indeed, in rodents the peripheral application of polysulfated anions prevented the development of prion disease, or significantly prolonged the incubation time (40-42). In addition, the intraventricular infusion of pentosan polysulfate, a sulfated GAG, significantly prolonged the incubation time and reduced neurodegenerative changes and infectivity in a mouse model of prion disease (15). Subsequently, in several case reports and 2 observational studies in small patient cohorts, researchers observed a possible extension of survival in patients with long-term intraventricular treatment with pentosan polysulfate (14,43-45). For tau, we have previously determined that heparin inhibits cellular uptake in vitro, and a polysulfated dextran derivative reduced neuronal tau uptake in mice (1). We and others have found that the interaction of tau aggregates with heparin derivatives depend on critical sulfate moieties on the sugar chain such as 6-O-sulfation (2,46) and N-sulfation (2). A recent study concluded that heparin-like short oligosaccharides limit the cellular uptake of tau oligomers, and reduce toxicity in cell culture (11). Taken together, the data from cell and animal studies suggested that heparin derivatives could represent a valid therapeutic approach to block the cellular uptake of tau and other prions, and thus prevent the spread of pathology.

[000104] Consequently, the inventors synthesized a heparinoid, SN7-13, with very low molecular weight (2.5-8 kDa), low charge, and without anticoagulation activity. It was created in a novel fashion based on aliphatic linkage of pentasaccharide units (Figure 2). SN7-13 has high affinity for tau, blocks tau aggregate uptake, and prevents replication in a HEK293T cells and primary neurons (Figure 12, 13). This suggests that a compound of this class could have therapeutic potential.

[000105] In summary, the inventors have created a novel molecule class that exhibits many desirable characteristics as inhibitors of tau propagation. SN7-13 provides a foundation for designing heparinoid derivatives for the treatment of neurodegenerative disorders and other diseases such as cancer, infections and inflammatory diseases that might respond to specific inhibition of protein binding to HSPGs (47). Example 2

In Vivo Assessment of Novel Heparinoid Like Compound, SN7-13

[000106] The following study was conducted in order to assess the in vivo activity of SN7- 13.

Materials and Methods Animal Model And Maintenance

[000107] Animal handling and maintenance was done as previously described (48, 50). Transgenic mice expressing 4R1N P301S human tau under the murine prion promoter (33) were obtained from Jackson Laboratory, and maintained on a B6/C3 background. Transgenic mice and wild-type littermates were housed under a 12-hour light/dark cycle, and were provided food and water ad libitum. For all experiments, conditions were gender-matched. All experiments involving animals were approved by the University of Texas Southwestern Medical Center institutional animal care and use committee.

Toxicity Studies In Mice

[000108] Toxicity study 1 : A total of 6 female P301S tau transgenic mice at age 2.5 mos were anesthetized with isoflurane and kept at 37°C throughout the inoculation. 10 pL gas-tight Flamilton syringes were used to inject 2 mΐ of compound at a rate of 0.2 mT per min in the left hippocampus (bregma: -2.5 mm posterior, -2 mm lateral, -1.8 mm ventral). Three (3) different compound concentrations (0.1 mg/ml, 0.33 mg/ml and 10 mg/ml) were injected in n=2 mice each. The mice were assessed clinically and did not observe any abnormal behavior. The mice were sacrificed after 24 hrs, and their brains were assessed for macroscopic signs of hemorrhage. No clinical or macroscopic signs of toxicity or hemorrhage were observed in this study.

[000109] Toxicity study 2: A total of 12 P301S tau transgenic mice were divided in into 4 groups with 3 animals each (2 females and 1 male): Group 1 = PBS, group 2 = SN7-13 at 1 mg/ml, group 3 = SN7-13 at 3 mg/ml, group 4 = SN7-13 at 10 mg/ml. At age 2.5 month, mini- osmotic pumps (Alzet, Model 2004) were implanted into the right ventricle for all 12 mice, filled with PBS or compound as indicated above. The pumps deliver at a constant rate of ~25 mΐ/hr for a duration of 28 days. In this time frame clinical signs of toxicity were observed. The mice were sacrificed on day 28 post-implantation. The brains did not show any macroscopic signs of toxicity. Tau pathology was analyzed by AT8 staining and seeding assay experiments as described above, and no differences were observed between controls (PBS) and experimental animals (SN7-13). Also, no dose-dependent effects in the mice receiving SN7- 13 were observed.

Animal Tissue Collection

[000110] The mice were anesthetized with isoflurane and perfused with chilled PBS + 0.03% heparin. Brains were post- fixed in 4% PFA overnight at 4°C and placed in 30% sucrose in PBS until further use.

Histology

[000111] Flistology was performed as described previously (48). Brains were sectioned at 50 pm with a freezing microtome. Slices were blocked for 1 hr with 5% milk in TBS with 0.25% Triton X-100 (blocking buffer). Brain slices were incubated with biotinylated AT8 antibody (1 :500, Thermo Scientific) overnight in blocking buffer at 4°C. Slices were then incubated with the VECTASTAIN Elite ABC Kit (Vector Labs) in TBS prepared according to the manufacturer protocol for 30 min, followed by DAB development using the DAB Peroxidase Substrate Kit with the optional nickel addition (V ector Labs). Slices were imaged using the Olympus Nanozoomer 2.0-lTT (Flamamatsu).

Results

SN7-13 Does Not Cause Toxicity In Mice

[000112] The inventors tested for toxicity of SN7-13 in PS 19 mice (33). First 2 pi of SN7-13 at 10 mg/ml was inoculated into the left hippocampus of 6 mice. The mice were killed the next day and the brains were tested for signs of hemorrhage. No clinical or macroscopic signs of toxicity or hemorrhage were observed. Next, 12 mice were implanted with intraventricular mini-osmotic pumps (Alzet) into the right ventricle and infused PBS (3 mice) or SN7-13 at 1, 3 or 10 mg/ml (3 mice each) for 28 days. No clinical or macroscopic signs of toxicity or hemorrhage observed. In addition, no change (increase/ decrease) of tau pathology (assessed by AT8 staining and tau seeding assay) was observed in animals that received SN7-13 in comparison to PBS.

[000113] While the present disclosure has been discussed in terms of certain embodiments, it should be appreciated that the present disclosure is not so limited. The embodiments are explained herein by way of example, and there are numerous modifications, variations and other embodiments that can be employed that would still be within the scope of the present disclosure.

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