WO2024035268A2 - Tau pathway modulators - Google Patents

Abstract

Pharmaceutical compositions and methods for treating tauopathies are provided.

Description

TAU PATHWAY MODULATORS

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional patent application 63/396,855 filed August 10, 2022, the entire contents of which are incorporated by reference herein.

TECHNICAL FIELD

[0002] This disclosure relates to modulators of the tau pathway.

BACKGROUND

[0003] Alzheimer’s disease (AD) is a progressive neurodegenerative disease and considered the predominant cause of dementia worldwide. The pathological hallmarks of AD are accumulated amyloid p plaques and the neurofibrillary tangles. While the amyloid plaques may play a key role in AD pathogenesis, clinicopathologic correlation studies indicate that the severity of the cognitive impairment correlates best with the burden of neurofibrillary tangles.

[0004] In addition, multiple neurodegenerative disease termed tauopathies, such as Progressive Supranuclear Palsy (PSP) and Frontotemporal dementia (FTD), linked to mutations in tau protein also exhibit tangle pathology. It is established that hyperphosphorylation of tau is responsible for the neurofibrillary lesions found in these conditions. Studies have shown that reduction in wild-type tau prevents Ap-dependent behavioral and cognitive deficits, suggesting that therapeutic interventions that alter the levels of tau may be beneficial. The transcription factor specificity protein 1 (Sp1) is essential for the regulation of tau, and cyclin dependent kinase 5 (CDK5). We have provided convincing evidence that either silencing of the Sp1 gene using small interfering RNA, or treatment of animals with Tolfenamic Acid (TA) lowers the expression of AD- related Sp1 target genes. Indeed, TA can reduce the production of tau, phosphotau (ptau), and CDK5.

[0005] In addition to its action on molecular intermediates, TA also modifies the ensuing pathology and provides cognitive improvement in Tau transgenic mice. Recently, we have obtained evidence that TA interacts directly with Sp1 and lowers its sequencespecific DNA binding. TA (CLOTAM® Rapid) is currently used in Europe and other countries to treat symptoms of migraine headaches. TA represents a class of drugs that can cross the blood brain barrier (BBB) and impact tau pathology by its unique ability to selectively act at the DNA-binding domain of cerebral Sp1. TA is the only NSAID that interacts and lowers Sp1 levels.

SUMMARY

[0006] Disclosed embodiments comprise compositions and methods for downregulating tau activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, as will occur to one having ordinary skill in the art and having the benefit of this disclosure.

[0008] FIG. 1 is a proposed mechanism for TA-induced down-regulation of tau and innovative synthetic scheme of TA analogs. TA promotes the degradation of the transcription factor Sp1 that results in a decrease in tau and CDK5 transcription, and, thus, lowers the levels of total tau and phosphorylated tau, ultimately, reducing one of the pathological hallmarks of tauopathies.

[0009] FIG. 2 shows cytotoxicity screening of a series of TA analogs. Differentiated Neuroblastoma cells were exposed to analogs for 48 h. The diagrams on the left show the status of the cells and the graphs depict cell viability using the MTS method after exposure to analogs at a dose range from 5-100 pM for 48 h.

[0010] FIGS. 3A-3F show TA, TN3 and TN7 lower AD-related proteins. Differentiated SH-SY5Y cells were exposed to 25 pM of lead acetate for 48 hours and were subsequently treated with 25 pM of TA, TN3 or TN7 for 72 hours. Western blot analysis was performed on total protein lysates. (FIGS. 3A-3C ) Blots and quantification of APP, tau and GSK3p. Protein levels were normalized to GAPDH. (FIGS. 3D-3F) Blot and quantification of APP. The impact of TA exposure on both tau and ptau is shown. Protein levels were normalized to GAPDH. Data are expressed as ± S.E.M. and statistical significance was determined using the one-way ANOVA followed by Dunnett’s for multiple comparisons, n was 3 for each treatment group. (*P <0.5, **P < 0.01 , *** P < 0.001 , ****P < 0.0001 ). * Denotes comparison to DMSO control and # denotes comparison to TA.

[0011] FIGS. 4A-4C show TA and analogs altered SP1 protein levels and DNA binding but did not change Sp1 mRNA expression Differentiated SH-SY5Y cells were exposed to 25 pM lead acetate for 48 hours to induce SP1 expression. Subsequently, the cells were treated with TA, TN3 and TN7 at 25 pM for 72 hours and samples were collected for RT-qPCR or western blot analysis. (FIGS. 4A-4B ) Blots and quantification of SP1 protein. (FIG. 4C) Quantification of Sp1 mRNA expression. Protein and mRNA expression were normalized to GAPDH. Protein lysates were also collected for EMSA analysis using treated cells and biotinylated SP1 oligos (Right Panel), ‘-ve’ indicates probe only negative control ‘+ve’ indicates labelled probe and total protein lysate positive control * indicates labelled probe, total protein lysates and unlabeled probe. Triangle indicates increased concentrations (0, .01 , 1 , 5 and 25 pM). Data are expressed as ± S.E.M. and statistical significance was determined using the one-way ANOVA followed by Dunnett’s for multiple comparisons, n was 3 each treatment group. (*P <0.5, **P < 0.01).

[0012] FIG. 5 shows TA and TN3 reduce SP1 bound to the APP promoter region. Differentiated SH-SY5Y cells were exposed to 25 pM lead acetate for 48 hours to induce SP1 expression. Subsequently, the cells were treated with TA, TN3 and TN7 at 25 pM for 72 hours. Then, fixed samples were collected for ChIP analysis using SP1 to pull down DNA. (A) ChlP-qPCR analysis of APP. Data are expressed as ± S.E.M. and statistical significance was determined using the one-way ANOVA followed by Dunnett’s for multiple comparisons, n was between 4 and 6 for each treatment group. (*P <0.5).

[0013] FIG. 6 shows proteins affected by treatment with TA and its analogs. Global proteomic analysis was conducted using SWATH-MS DIA proteomics. The data were analyzed and visualized using spectronaut, Qiagen IPA and R. Each Venn diagram shows the number of modulated proteins of each treatment group and overlapping groups compared to the control group.

[0014] FIG. 7. Dementia demographics in Qatar. People above 65 years represented 1 .5 percent (43,000) of the total population Qatar This number is expected to increase to 14.2 percent (546,000) by 2050. There are an estimated 4,400 persons with dementia in Qatar. This number is expected to rise to over 40,000 by 2050.

[0015] FIG. 8. AD biomarkers. Preclinical AD, Onset of cognitive impairment, Symptomatic AD.

[0016] FIG. 9. Tau pathology. Hyperphosphorylation of tau is responsible for the neurofibrillary lesions.

[0017] FIG. 10. TA and its analogs. Conformations after modifications of selected functional group were superimposed on a prototype TA conformation. The tendency of each compound to bind zinc ion in gas phase was studied. Complete geometry optimizations were carried out in presence of zinc allowing the compound to reorganize to capture the ion in the most efficient way. The overall binding affinity was then viewed as a combination of stabilization gained in binding and the loss in conformational rearrangement required for efficient capture of the ion.

[0018] FIG. 11. TA and its analogs. Cytotoxicity screen. TN3 and TN7 showed better safety profiles than TA 72h after treatment. [0019] FIGS. 12A-12C. TA and its analogs- Sp1 ChlP_Seq. FIG. 12A shows plots for DMSO, TA, TN3 and TN7. FIGS. 12B-12C show Sp1 ChlP_Seq data.

[0020] FIGS. 13A-13C. TA and its analogs. Transcriptomics (mRNA_Seq). Volcano plots of pairwise differential gene expressions. Volcano plots of TA (FIG. 13A), TN3 (FIG. 13B) and TN7 (FIG. 13C) with DMSO.

[0021] FIG. 14. TA and its analogs. Transcriptomics (mRNA_Seq).

[0022] FIG. 15. TA and its analogs. Study design.

[0023] FIG. 16. Tolfenamic acid (TN1 , kCMZ605) plasma and terminal brain exposure.

[0024] FIGS. 17A-17C. TA and its analogs. Brain penetration. TN3 and TN7 can cross the blood-brain barrier. Concentration vs time plots for TA (FIG. 17A), TN3 (FIG.

17B) and TN7 (FIG. 17C)

[0025] FIGS. 18A-18C. TA and its analogs. Transcriptomics (in vivo). TA vs WT (FIG. 18A); TN3 vs WT (FIG. 18B); TN7 vs WT (FIG. 18C).

[0026] FIG. 19. TA and its analogs. Transcriptomics (in vivo).

[0027] FIGS. 20A-20B provide gene and detail information. TN3 and Tn7 target neurodegenerative pathways in the CNS.

[0028] FIG. 21. TA and its analogs. Proteomics (in vivo).

[0029] FIGS. 22A-22D show overview of synthetic strategy, lead compounds and their safety.

[0030] FIGS. 23A-23H show mode of action of compounds acting at the gene level; at transcription start sites to interfere with SP1 binding.

[0031] FIGS. 24A-24D show compounds alter expression of genes that are primarily SP1 targets. [0032] FIGS. 25A-25E show final outcome of interfering at the gene level on the protein products by interrogating the whole proteome. The bar graph displays the outcomes on specific proteins involved in neurodegenerative diseases such as Alzheimer’s.

DETAILED DESCRIPTION

[0033] Dicslosed embodiments comprise compositions and methods for treating tauopathies, for example using TA analogs and derivatives. The structure of tolfenamic acid is shown below:

tolfenamic acid

[0034] Aspects of the present disclosure are directed to pharmaceutical compositions that include a compound of Formula I or Formula II,

Formula I Formula II or a pharmaceutically acceptable carrier, wherein Y is S, NH, 0 or CH2; R¹ is H or C1-6 alky; and R², R³, R⁴, R⁵, R⁶, and Z are each independently H, F, Cl, Br, NO2, CF3, CN, C1- 6 alky, or OCnH2n+i, wherein n is 1 to 6.

[0035] In some embodiments, the pharmaceutical composition includes the compound of Formula I, wherein Y is S; R¹, R³, R⁴, R⁵, R⁶ and Zare H; and R² is CN.

[0036] In some embodiments, the pharmaceutical composition includes the compound of Formula I, wherein Y is S; R¹, R² and Z are H; and one of R³, R⁴, R⁵, or R⁶ is CN.

[0037] In some embodiments, the pharmaceutical composition includes the compound of Formula II, wherein Y is NH; R¹, R², R³, R⁴, R⁶ and Z are H; and R⁵ is CF3.

[0038] In some embodiments, the pharmaceutical composition includes the compound of Formula II, wherein Y is NH; R¹, R⁵ and Z are H; and one of R², R³, R⁴, or

R⁶ is CF3.

[0039] In some embodiments, the the compound of Formula I or the compound of

Formula II includes

or a pharmaceutically acceptable salt thereof. [0040] The names of the foregoing compounds are disclosed in Table 1 .

[0041] Table 1

[0042] Another aspect of the present disclosure is directed to methods of downregulating tau protein expression or activity in a patient in need thereof. The method includes administering to the patient a therapeutically effective amount of a compound of Formula I or Formula II,

Formula I Formula II or a pharmaceutically acceptable carrier, wherein Y is S, NH, 0 or CH2; R¹ is H or C1-6 alky; and R², R³, R⁴, R⁵, R⁶, and Z are each independently H, F, Cl, Br, NO2, CF3, CN, C1- 6 alky, or OCnH2n+i, wherein n is 1 to 6.

[0043] In some embodiments, the compound is Formula I, wherein Y is S; R¹, R³, R⁴, R⁵, R⁶ and Zare H; and R² is CN.

[0044] In some embodiments, the compound is Formula I, wherein Y is S; R¹, R² and Z are H; and one of R³, R⁴, R⁵, or R⁶ is CN.

[0045] In some embodiments, the compound is Formula II, wherein Y is NH; R¹, R², R³, R⁴, R⁶ and Z are H; and R⁵ is CF₃.

[0046] In some embodiments, the compound is Formula II, wherein Y is NH; R¹, R⁵ and Zare H; and one of R², R³, R⁴, or R⁶ is CF3.

[0047] In some embodiments, the compound of Formula I or the compound of

Formula II includes

[0048] Another aspect of the present disclosure is directed to methods of treating a neurodegenerative disease in a patient in need thereof. The method includes administering to the patient a therapeutically effective amount of a compound of Formula I or Formula II,

Formula I Formula II or a pharmaceutically acceptable carrier, wherein Y is S, NH, 0 or CH2; R¹ is H or C1-6 alky; and R², R³, R⁴, R⁵, R⁶, and Z are each independently H, F, Cl, Br, NO2, CF3, CN, C1- 6 alky, or OCnH2n+i, wherein n is 1 to 6.

[0049] In some embodiments, the compound is Formula I, wherein Y is S; R¹, R³, R⁴, R⁵, R⁶ and Zare H; and R² is CN.

[0050] In some embodiments, the compound is Formula I, wherein Y is S; R¹, R² and Z are H; and one of R³, R⁴, R⁵, or R⁶ is CN.

[0051] In some embodiments, the compound is Formula II, wherein Y is NH; R¹, R², R³, R⁴, R⁶ and Z are H; and R⁵ is CF₃.

[0052] In some embodiments, the compound is Formula II, wherein Y is NH; R¹, R⁵ and Zare H; and one of R², R³, R⁴, or R⁶ is CF3. [0053] In some embodiments, the compound of Formula I or the compound of Formula II includes

or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the neurodegenerative disease includes a tauopathy.

[0055] In some embodiments, the neurodegenerative disease includes Alzheimer’s

Disease.

Definitions:

[0056] “A” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0057] “Comprise,” “comprising,” “include,” “including,” “have,” and “having” are used in the inclusive, open sense, meaning that additional elements may be included. The terms “such as”, “e.g.”, as used herein are non-limiting and are for illustrative purposes only. “Including” and “including but not limited to” are used interchangeably.

[0058] “In vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell culture. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment. [0059] “Or” as used herein should be understood to mean “and/or”, unless the context clearly indicates otherwise.

[0060] “Patient,” “subject,” or “host” to be treated by the subject method can mean either a human or non-human animal, such as a mammal, a fish, a bird, a reptile, or an amphibian.

[0001] “Parenteral administration” and “administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, retro-orbital, intraocular, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0002] “Pharmaceutically acceptable” or “therapeutically acceptable” refers to a substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not toxic to a patient

[0003] “Pharmaceutically acceptable carrier” is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non- pyrogenic. Exemplary materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0004] “Pharmaceutical composition” refers to a formulation containing the therapeutically active agent(s) described herein in a form suitable for administration to a subject. In embodiments, the pharmaceutical composition is in bulk or in unit dosage form. The quantity of active ingredient in a unit dose of composition is an effective amount and can be varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. In a preferred embodiment, the active ingredients are mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0005] The compositions or dosage forms may contain any one of the compounds and therapeutic agents described herein in the range of 0.005% to 100% with the balance made up from the suitable pharmaceutically acceptable excipients. The contemplated compositions may contain 0.001 %-100% of any one of the compounds and therapeutic agents provided herein, in one embodiment 0.1 -95%, in another embodiment 75-85%, in a further embodiment 20-80%, wherein the balance may be made up of any pharmaceutically acceptable excipient described herein, or any combination of these excipients.

[0006] In some embodiments, an effective amount of Tolfenamic Acid (TA) or an analog or derivative or a pharmaceutically acceptable salt thereof (and/or one or more of the other listed TA analogs or pharmaceutically acceptable salts thereof) can range, for example, from about 0.001 mg/kg to about 500 mg/kg (e.g., from about 0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 150 mg/kg; from about 0.01 mg/kg to about 100 mg/kg; from about 0.01 mg/kg to about 50 mg/kg; from about 0.01 mg/kg to about 10 mg/kg; from about 0.01 mg/kg to about 5 mg/kg; from about 0.01 mg/kg to about 1 mg/kg; from about 0.01 mg/kg to about 0.5 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 0. 1 mg/kg to about 200 mg/kg; from about 0. 1 mg/kg to about 150 mg/kg; from about 0. 1 mg/kg to about 100 mg/kg; from about 0.1 mg/kg to about 50 mg/kg; from about 0. 1 mg/kg to about 10 mg/kg; from about 0.1 mg/kg to about 5 mg/kg; from about 0.1 mg/kg to about 2 mg/kg; from about 0.1 mg/kg to about 1 mg/kg; or from about 0.1 mg/kg to about 0.5 mg/kg).

[0007] In some embodiments, an effective amount of Tolfenamic Acid (TA) or an analog or derivative or a pharmaceutically acceptable salt thereof (and/or one or more of the other listed TA analogs or pharmaceutically acceptable salts thereof) is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, or about 5 mg/kg.

[0008] The foregoing dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, thrice daily) or nondaily basis (e.g., every other day, every two days, every three days, once weekly, twice weekly, once every two weeks, once a month).

[0061] “Reducing”, “suppressing” and “inhibiting” have their commonly understood meaning of lessening or decreasing.

[0062] “Treatment” or “treating” refers to any therapeutic intervention in a mammal, for example a human or animal such as a companion animal, including: (i) prevention, that is, causing the clinical symptoms not to develop, e.g., preventing infection or inflammationfrom occurring and/or developing to a harmful state; (ii) inhibition, that is, arresting the development of clinical symptoms, e.g., stopping an ongoing infection so that the infection is eliminated completely or to the degree that it is no longer harmful; and/or (iii) relief, that is, causing the regression of clinical symptoms, e.g., causing a relief of fever and/or inflammation caused by or associated with a microbial infection. T reatment can comprise multiple administrations of compositions disclosed herein.

[0063] Tangles, which are deposits of hyperphosphorylated tau, are found in neurodegenerative disorders that are referred to as tauopathies, of which, AD is the most common. Others include: Guam Parkinsonism dementia complex, dementia pugilistica, corticobasal degeneration, Pick's disease, FTD, Parkinsonism linked to chromosome 17 (FTDP-17), and PSP. The microtubule associated protein tau (MAPT) was first isolated and identified as a protein necessary for microtubule assembly. The normal function of tau is to stabilize microtubules, and the exact cause of its aggregation is unknown, but it has been observed that tau hyperphosphorylation reduces its binding affinity to microtubules and is suspected to play a role in its aggregation. Moreover, it suppresses microtubule assembly; suggesting that phosphorylation regulates the functions of tau. Mutations in the tau gene have been found in FTDP-17 where only tau aggregates, not (Ap) plaques, are the characteristic deposits. In addition, tau mutations are responsible for 5% of all FTD cases. Neurofibrillary degeneration in the absence of Ap is seen in several tauopathies with neocortical lesions, clinically characterized by dementia. Furthermore, neurofibrillary pathology correlates best with the presence of dementia in humans. To date, no drug has been found to stop the progression of AD and there is no FDA approved drug to treat other tauopathies. All available medications are prescribed as symptomatic or adjuvant therapy. While many AD drug candidates have been tested for their ability to interrupt the amyloid pathway, little focus has been given to drugs that could interfere with the phosphorylation of tau or reducing its levels, and, hence, alter the formation of tangles. In an APP transgenic mouse model, lowering of tau levels improved cognitive performance and prolonged the lifespan of animals, although, amyloid deposits were not altered. It was further reported that lowering of soluble hyperphosphorylated tau rather than the insoluble tangles correlates with cognitive improvement. In fact, in a neurodegeneration mouse model, tau inhibition recovered memory function even though the buildup of tangles continued suggesting that tangles, alone, are not responsible for cognitive dysfunction. It is becoming clear that research strategies and therapeutic protocols that focus on elimination of the toxic “end products” of AD and tauopathies are unlikely to produce a significant disease modification. In all likelihood, considerable irreparable damage has occurred before the point when the toxic proteins accumulate in plaques and tangles. It is vitally important that we identify and target events that are “upstream” of the final toxic products, when the functional damage may be reversible or even preventable.

[0064] We explored the therapeutic potential of TA in modifying disease processes in a transgenic animal model that carries the human tau gene (hTau). Behavioral tests demonstrated the efficacy of TA in improving spatial learning deficits and memory impairments in young and aged hTau mice. Western blot analysis of the hTau protein revealed reduction in total tau and site-specific hyperphosphorylation of tau in response to TA administration. Immunohistochemical analysis for phosphorylated tau protein revealed a reduction in staining in the frontal cortex, hippocampus, and striatum in animals treated with TA. TA, thus, holds the potential as a disease-modifying agent for the treatment of tauopathies. TA (CLOTAM® Rapid) is currently used in Europe and other countries to treat symptoms of migraine headaches and is available in 200 mg tablet form. TA represents a class of mechanism-based drugs for the treatment of AD that could interfere with AD pathogenesis through pathways independent of its anti-inflammatory properties.

[0065] In animal studies, TA showed the lowest gastro-ulcerogenicity among 11 tested NSAIDs. We have also found epidemiologic evidence, in the literature, with regards to TA and cardiovascular effects, suggesting that TA imparts lower risk than many NSAIDS. The LD50 is between 200-1000 mg/kg/bw, depending on the animal species and route of administration. Oral administration of TA to mice did not cause any abnormal changes in behavior, activity or appearance. TA did not produce any mutations in vitro or in vivo and was non-carcinogenic in mice and rats after 80 and 104 weeks, respectively, of oral treatment with 15, 30 and 60 mg/kg/day. TA had no major effect on respiration, heart rate and blood pressure in dogs, rabbits and rats after intravenous doses reaching 10 mg/kg. In humans, TA administered daily at 600 mg for 6 months for rheumatoid arthritis was safe and has made it acceptable for long term use. In this proposal, we plan to investigate derivative of TA with better safety and higher efficacy, and examine their specific mechanism of action, and off-target effects.

[0066] TA represents a class of drugs that can impact tau pathology by its unique ability to act at the DNA-binding domain of Sp1. TA is the only NSAID that interacts and lowers Sp1 levels. Sp1 is a transcription factor involved in AD pathology, whose mRNA and protein levels are elevated within the frontal cortex of AD patients as well as in animal models with AD-like pathology. In addition, Sp1 regulates the expression of tau, therefore, mutations on the Sp1 binding regions of the tau promoter decrease tau expression. Sp1 also regulates the transcription of CDK5 activators p39 and p35; Sp1 binding motifs were found on the promoter regions of CDK5, p35 and p39. CDK5, a member of the cyclin- dependent kinase family, is responsible for the phosphorylation of tau on sites that are unusually hyperphosphorylated in AD. Generally, only a minority of genes (<10%) possess multiple GGGCGG sequences in their promoter region. In other words, 90% of all genes would not be affected by the multitude of zinc finger transcription factors that target these sequences and even a smaller set of genes will be expected to be targeted by SP1. Therefore, targeting Sp1 is an ideal approach to lower tau levels, and such reduction is likely to impact posttranslational modifications of tau, thereby providing a mechanistic approach to reduce the pathological features of neurodegenerative diseases and enhancing cognitive improvement (FIG. 1).

[0067] Cytotoxicity screen of analogs

[0068] Published in vivo studies in animal models show that TA reduces biomarker levels and immuno-reactivity associated with tauopathy. This further substantiates the ability of TA to interfere in pathways associated with neurodegeneration. In the following study, we synthesized a series of TA analogs with a strategy to minimize their COX- related activity and maximize their action through the Sp1 pathway (FIG. 2). After multiple screens, we advanced eight analogs from a series of compounds for cytotoxicity testing and those that exhibited a better safety profile better than TA and had lower impact on cell viability at the dose range used. TN3 and TN7 appeared to exhibit the best safety profile and, therefore, we selected them as lead compounds for further testing.

[0069] Promising in vitro outcomes of the lead analogs

[0070] Primed SH-SY5Y cells that were treated with TA, TN3 and TN7 for 72 hours at 25 pM the optimal dose and time selected from range finding studies. We found that TN3 and TN7 lowered total tau protein levels compared to the control, while TA had a lesser effect (FIG. 3B). TN7 lowered total tau protein compared to TA as well and significantly decreased GSK3p protein levels (CDK5 may be analyzed in the proposed study) compared to TA and the control (FIGS. 3B-3C). TN3 and TN7 outperformed TA on lowering APP protein levels (FIG. 3E). [0071] Preliminary dose-response studies show that TA reduces both the tau protein and its phosphorylated forms, in a dose-dependent manner. Furthermore, the reduction in the phosphorylation of tau appear to mirror the reductions in total tau, suggesting that these phosphorylation changes may be related to the availability of tau and its biosynthetic pathway.

[0072] TA and analogs altered SP1 protein levels and DNA binding but did not change Sp1 mRN A expression

[0073] Primed SH-SY5Y cells were treated with TA, TN3 and TN7 for 72 hours at 25 pM. TN3 and TN7 significantly decreased SP1 protein levels compared to the control, however, TA, TN3 and TN7 did not significantly affect the mRNA expression of SP1 (FIGS. 4A-4C). TA, TN3 and TN7 decreased SP1-DNA binding using EMSA analysis. TA decreased SP1 -DNA binding at 25 pM (FIG. 5), while TN3 and TN7 appeared to decrease SP1 binding at 5 and 25 pM.

[0074] The functional impact of decreased Sp1 DNA binding on gene expression

[0075] To explore whether the impact of TA and its analogs on Sp1 DNA binding had a functional effect, we used the ChlP-qPCR method which employs an antibody directed against SP1 to pull down promoter regions of putative AD-related target genes. This demonstrated that treatment with TA or TN3 showed a strong trend (p=0.0547, 0.0733, respectively) of decreased SP1-DNA binding to the APP promoter region (FIG. 6).

[0076] These data are consistent with the report that siRNA silencing and depletion of SP1 significantly reduced APP promoter responsiveness. Future work will examine the impact on the btau promoter.

[0077] Global proteomic analysis to determine the scope of protein changes following exposure with TA and analogs

[0078] In this study, we analyzed the on-target and off-target proteomic profiles of TA, TN3 and TN7 in an in vitro model of AD using differentiated SH-SY5Y cells which were primed with lead acetate to upregulate SP1 and tau expression. Treatment with TA, TN3 or TN7 resulted in a global proteomic shift compared to the control with overlapping proteomic changes (FIG. 6). The venn diagram demonstrated that approximately 14.54% of perturbed proteins were shared between all three treatment groups with another 35.1 % of differentially expressed proteins shared between 2 treatment groups. These findings were expected as TN3 and TN7 were synthesized by using TA as a scaffold structure. Interestingly, TN3 and TN7 shared the greatest number of differentially expressed proteins compared to their overlaps with TA which may be indicative that TN3 and TN7 have similar pharmacodynamics.

[0079] Compositions

[0080] Disclosed embodiments comprise compositions for lowering or downregulating tau protein expression, activity, or both. In embodiments, disclosed compositions display improved safety profiles as compared to treatments such as, for example, Tolfenamic Acid (TA).

[0081] For example, in embodiments, such compositions can comprise TA, and analogs and derivatives thereof. Disclosed compositions can comprise at least one of TA and analogs and derivatives thereof. Disclosed compositions can comprise more than one TA or analog or derivative thereof.

[0082] Methods of Use

[0083] Disclosed embodiments comprise methods of use of disclosed compositions. For example, in embodiments, disclosed compositions are formulated in a pharmaceuticaccly acceptable carrier.

[0084] In embodiments, administration can be performed via any appropriate method, such as by injection.

EXAMPLES [0085] Example 1 - Investigate the ability of analogs with a better safety profile than TA to lower thebiochemical levels of the tau mRNA, protein, p-tau, Sp1, and COX2 in vitro

[0086] We have selected compounds with better safety profile than TA and may measure their impact on biomarkers associated with the tau pathway, Sp1 , and COX2, using iPSCs.

[0087] Generation, culture and maintenance of induced pluripotent stem cells (iPSCs)

[0088] The iPSCs may be generated from the somatic cells by transduction with CytoTune-iPS 2.0 Sendai reprogramming kit (ThermoFisher Scientific). After 3 days of reprogramming, the cells may be seeded on Matrigel-coated plates without cytokines. At day 7, half of the medium may be replaced with ReproTeSR medium (Stem Cell Technologies), and the medium may be gradually completely changed to ReproTeSR. The emerged iPSCs colonies may be manually picked, expanded, and maintained in mTESR-1 medium (Stem Cell Technologies) supplemented with 1 % penicillinstreptomycin (Gibco) on Matrigel-coated (BD Biosciences) plates.

[0089] Neural precursor cells derivation and culture (NPCs) and treatment with TA and analogs

[0090] The established undifferentiated iPSCs of the generated clones may be dissociated using TrypLE™ (Gibco) and cultured on ultralow- attachment plates in mTeSR media with 10 pM Rock inhibitor Y27632 (Stemgent) for the 24 hours. The newly formed aggregate which are named Embryoid bodies (EBs) may be cultured in suspension for 4 days in differentiation Knockout DMEM medium which is composed of KO-DMEM medium, 20% Knockout serum replacement, 1 % pen/strep, 1 % L- Glutamine and 1X non-essential amino acid NEAA, and 0.1 mM p- mercaptoethanol supplemented by 10uM SB431542, 2uM Dorsomorphin and 10 ng/mL b-FGF. At day 4, the medium may be changed to neural induction medium (DMEM/F12, 1X N2, 1% L- Glutamine, 1X NEAA, 0.1 mM p-mercaptoethanol and 2ug/mL Heparin) supplemented with 10uM SB431542, 2uM Dorsomorphin and 10 ng/mL b-FGF. At day 6, the 3D aggregates may be plated on polyornithine/laminin coated plates and may be cultured in neural induction medium supplemented with 2uM Dorsomorphin and 10 ng/mL b-FGF for 10 days.

[0091] The formed neural rosettes may be manually harvested and replated on polyornithine/laminin coated plates with neural induction medium supplemented 10 ng/mL b-FGF for one more week (7 days). The purified newly formed rosettes may be manually collected and dissociated to single neural precursor cells (NPCs) using Accutase cell dissociation reagent (Stem cell technology). The generated NPCs may be cultured and maintained in the neural precursor medium (DMEM/F12, 1X N2, 1X B27 without Vit A, 1 ug/ mL Laminin, 1 % L- Glutamine, 1X NEAA, 0.1 mM p-mercaptoethanol and 2ug/mL Heparin) supplemented 10 ng/mL b-FGF. The NPCs can be passaged every 3-4 days (1 :3 or 1 :4) and can be cryopreserved for long-term use. The neural precursor cells were plated at a density of 50,000 cells per well in complete neural differentiation media (Neurobasal, 1X N2, 1X B27 without Vit A, 1X L-Glutamine, 1X non-essential amino acids, and 1 g/ml Laminin) supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml glial cell-derived neurotrophic factor (GDNF), 10ng/ml insulin growth factor (IGF-1 ), 1 pM dibutyryl cyclic adenosine monophosphate (cAMP), and 200 ng/ml Ascorbic acid. The media was changed every second day for 6-8 weeks until getting the mature functional neurons. Then, various concentrations of TA or analogs may be incubated with the cells. The cells may be harvested at 24 h, 48 h, and 72 h, with media being changed every three days.

[0092] Protein extraction and Western blotting

[0093] Total protein extraction from cell lysates may be performed following the procedure described previously. Protein concentration may be determined using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) and extracts may be stored at -80 °C. Western blotting and quantification may follow procedures from previously published studies. Immunoblotting may be carried out following overnight exposure to the following antibodies listed in Table 2. [0094] Table 2 shows Antibody Specifications- the blots may be exposed for 1 h to IRDyeR 680LT infrared dye (LI-COR Biotechnology, Lincoln, NE, USA) goat antimouse/ goat anti-rabbit diluted at 10: 10,000 after washing with Tris-buffered saline (TBS) and TBS Tween-20 (TBST). Finally, OdysseyR Infrared Imaging System (Li-Cor, NE) may be used to detect and quantify infrared signal of Western Blot bands. Loading of the samples may be assessed by staining the gels with Bio-safe Coomassie blue stain (Bio-Rad, Hercules, CA, USA) following the transfer to PVDF membranes.

[0095] Table 2

[0096] Total RNA isolation, synthesis of complementary DNA, and real-time polymerase chain reaction

[0097] Total RNA extraction from the SH5Y cells and cDNA synthesis may be carried out. We may be using the following murine primers Sp1 F: 5-TCA TAC CAG GTG CAA ACC AA-3 (SEQ ID NO:1); R: 5-AGG TGA TGT TCC CAT TCA GG-3 (SEQ ID NO:2); CDK5F: 5-TGG TGA AGC AGG CAT CTG AG-3 (SEQ ID N0:3); R: 5-CCA TTG CAG CTG TCG AAA TA-3 (SEQ ID N0:4);GAPDH F: 5-TGG TGA AGC AGG CAT CTG AG-3 (SEQ ID N0:5); R: 5-TGC TGT TGA AGT CGC AGG AG-3 (SEQ ID N0:6). The following human primer may be used TGA GG-3. ABI PRISM 7500 machine (Applied Biosystems, Carlsbad, CA, USA) with Sequence Detection Software (SDS) version 1.3 may be used for amplification and expressions may be reported relative to GAPDH mRNA with 2-AACT method.

[0098] COX1/2 activity Assay

[0099] The COX activity assays may be used as a counter screen to select the candidates with optimal Sp1 DNA binding and Tau promoter activity and minimal COX1/2 activity. The COX Activity Assay utilizes the peroxidase component of cyclo-oxygenases. The peroxidase activity may be assayed calorimetrically by monitoring the appearance of oxidized A/, A/, A/', A/'-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. The assay can be used to measure COX activity in cell lysates, tissue homogenates, and purified enzyme preparations. The assay includes COX-1 and COX-2 specific inhibitors to distinguish between the two isozymes (Cayman Chemicals).

[00100] Statistical treatment

[00101] One-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test may be used to compare the effects of various treatments versus the control group. Graph pad prism 7.0 computer software (La Jolla, CA, USA) may be used for analysis, P<0.05 may be considered significant.

[00102] Expected Outcomes

[00103] Previous studies have shown the ability of TA to lower AD related biomarkers in hTau transgenic mouse model independently from COX-2 pathway. Our preliminary studies, also, confirm the same in vitro, in a dose-dependent manner. We expect to see that changes in protein biomarkers may mirror those of their mRNA profile, substantiating that these compounds are acting via a transcriptional pathway. While the Sp1 protein may be reduced in the absence of any changes in its mRNA levels. We expect less COX activity of the analogs compared to TA. We may face issues where the mRNA profiles do not mirror their corresponding protein products. We may also face issues related to establishing dose-response relationships at the lower nanomolar concentrations.

[00104] Example 2 - Testing of lead analogs for mode of action and target engagement.

[00105] Here we will be testing our hypothesis that TA acts via the Sp1 mechanism using iPSCs. Lead analogs may be tested for their ability to disrupt Sp1 DNA binding, affect SP1 driven promoter activity of AD-related genes, and their ability to engage Sp1 as a target. ChiP-seq may be used to identify SP1 target genes and gene editing methodology may also be employed to knockout Sp1 from these cells and define the cellular response in the absence of this molecular mediator.

[00106] Cell culture: See above.

[00107] Chromatin Immunoprecipitation (ChlP-qPCR):

[00108] Cells were prepared for ChIP reactions and all reactions were conducted using the SimpleChIP Plus Chromatin Immunoprecipitation kit (Cat.9005, Cell Signaling Technology, Danvers, MA) according to the manufacturer’s protocol. Briefly, cells were crosslinked using 37% formaldehyde (Cat. F8775, Sigma-Aldrich, St. Louis, MO) at a 1 % final concentration in 20 ml of complete media for 1 minutes at room temperature. 2 ml of 10X glycine was added to each flask of cells and incubated at room temperature for 5 minutes. Media was removed and cells were washed twice with 1X PBS. Then, cells were scraped using ice-cold PBS supplemented with the protease-inhibitor included in the kit.

[00109] Afterwards, cells were centrifuged at 2000 g for 5 minutes at 4oC. Nuclei prep was conducted using manufacturer’s protocol with minor modifications. Nuclei were digested using 0.5 pl Micrococcal Nuclease per 4 x 106 cells at 37°C and tube was inverted every 3 minutes for 20 minutes before reaction was stopped using EDTA. Reaction was pelleted by centrifugation at 16000 g for 1 min at 4°C, supernatant was transferred to a new tube and lysate was sonicated 3 times for 15 seconds and allowed to rest 2-3 minutes between each sonication using the VWR Ultrasonic Homogenizer (Cat. 79193-588, VWR International, Radnor, PA).

[00110] Afterwards, lysates were clarified by centrifugation at 9400 g for 10 min at 4°C. Chromatin digestion was analysed using a 1 % agarose gel to electropherese the DNA with a 100 bp DNA marker. DNA was digested to be between 150-900 bp. DNA concentration was determined using the manufacturer’s recommendation. Chromatin immunoprecipitation reactions were carried out overnight (15-20 hours) at 4°C in rotation using 5-10 pg of digested chromatin, ChIP buffer supplemented with a protease inhibitor cocktail and 1.3 - 2.6 pg SP1 ChIP validated Rabbit mAb antibody (Cat. 9389, Cell Signaling Technology, Danvers, MA) or 1.3 - 2.6 pg of normal Rabbit IgG (Cat. 2729S, Cell Signaling Technologies, Danvers, MA) which was the negative control. Histone H3 XP Rabbit mAB was used as a positive control. ChlP-Grade Protein G beads were added to each reaction and incubated for 3 hours at 4°C with rotation. Protein G beads were pelleted using MagneSphere Technology Magnetic Separation Stands (Cat. Z5341 , Promega, Madison, Wl) then washed three times with a low salt wash then once with a high salt wash, pelleting the chromatin and removing supernatant after each wash, per manufacturer’s instructions.

[00111] To elute the DNA, the magnetic beads were suspended in 1X ChIP elution buffer, and samples were incubated at 65°C and 1200 rpm for 30 minutes in the Eppendorf ThermoMixer C (Cat. 5382000023, Eppendorf, Enfield, CT). Following elution, the magnetic beads were pelleted, the supernatant containing chromatin was transferred to a new tube, and the cross links were reversed by incubation at 65°C for 2 hours with proteinase k. Samples were purified using the supplied DNA purification kit according to the manufacturer’s protocol. qPCR reactions for promoter regions of interest were completed using the description above with supplied RLP3 primers serving as a positive control. Results were normalized to negative controls.

[00112] ChlP-seq

[00113] Relative enrichment of the target regions in the precipitated DNA fragments (described in “Chromatin Immunoprecipitation (ChlP-qPCR) section”) may be first analyzed by qPCR (Applied Biosystems) using Fast SYBER Green Master Mix (Applied Biosystems) for quality control of the ChlP. Once the ChIP QC was checked, the DNA library preparation may be performed using NEXTflex ChlP-Seq Barcodes 6 kit (Bioo Scientific, Austin TX) and then the quality of the library generated may be checked on an Agilent 2100 Bioanalyzer system followed by an accurate quantification using Qubit system.

[00114] Libraries that pass quality control may be pooled, clustered on a cBot platform, and sequenced on an Illumina HiSeq 4000 at a minimum of 25 million paired-end reads (2x75 bp) per sample.

[00115] CRISPR knock-out of SP1 in iPSCs

[00116] SP1 knock-out iPSCs may be generated to assess the impact of lead analogs in the presence or absence of SP1 in differentiated neurons. gRNAs targeting SP1 gene may be designed using online CRISPR Design Tool by Zhang Laboratory and ordered as synthetic sgRNA (Integrated DNA Technologies). Cells may be transfected with CRISPR- Cas9 (New England Biolabs, M0646M)/gRNA complexes targeting SP1 gene by electroporation using Nucleofector™ X KIT S (Lonza, Cat # V4XP-3032). Single-cell clones may be isolated from the cultured gene-edited pool and screened for SP1 knockout by RTPCR (BioRad) and immunoblotting (Abeam, ab13370) and confirmed by DNA Sequencing of on-target and offtarget regions.

[00117] Protein expression and purification

[00118] The plasmid containing Sp1 and any variants may be expressed in conventional E. coli systems using Innova Shaker/incubator as well as a BioFlow 115 fermenter. Various constructs may be expressed with affinity tags such as histidine and GST. The plasmids harboring genes may be amplified from cDNA libraries or may be acquired commercially from Addgene or Origene. mg quantities of proteins are usually obtained for most of the TFs expressed to date in the laboratory. Basic molecular biology procedures may be employed for the sub-cloning of these genes if required. For large- scale expression of recombinant constructs for structural and interaction studies, the plasmids may be transformed into bacteria E. coli strain Rosetta-2 or other strains. The expressed proteins may be purified using the Akta Xpress system combining various chromatographic techniques. Among the usual steps there is affinity purification using immobilized metals, ion exchange and gel filtration chromatography. Common chromatographic columns for these steps are Superdex 75 and 200 HR 10/30, HiLoad Superdex 200/75 16/60, MonoQ 5/5 MonoS 5/5, all from GE, as well as the metal affinity resin column.

[00119] Isothermal titration calorimetry (ITC)

[00120] Isothermal titration calorimetry is a direct and powerful technique for probing the thermodynamics of molecular interactions. It is the most popular and robust method for the determination of protein-protein or protein-ligand interactions and measures the heats of binding (AHbind) and binding free energy (AGbind) upon protein-protein/ ligand binding. It also provides the information about the stoichiometry (n) of the molecular interaction between the partners and their dissociation constant (KD) when the isotherm is fitted with the appropriate binding model. Affinity (Kd) of YAP/TEAD-DNA complexes may be availed through ITC. Other thermodynamic parameters such as DG and DS which give additional information regarding the type of interactions responsible for the binding (hydrophobic vs hydrophilic) may also be obtained through ITC. ITC experiments may be carried out by using a MicroCai Auto-iTC200 System (Malvern) Titrations may be carried out by injecting different concentration of partner protein solution into the ITC sample cell containing fixed amount compounds or vice versa. Control experiments may be performed to determine the heat of dilution and all ITC data may be corrected by subtracting the heats generated by titrating the partner protein or compound into buffer alone.

[00121] Protein Crystallization

[00122] Proteins may be concentrated to ~5 to ~10 mg/ml and crystallization trials may be set up using commercially available kits. The Mosquito robot may be used to enable sitting drop crystallization experiments. The Mosquito robot uses nL drops and thus a large number of crystallization attempts can be facilitated with minimal use of protein. Crystallization in complex with compounds may be attempted in similar manner. Various crystallization kits such as the Hampton and Qiagen screens may be used for the crystallization attempts. These initial screens may then be fine-tuned with a secondary screen bracketing the initial conditions. The detection of crystals may be monitored regularly by microscopy.

[00123] X-ray crystallography

[00124] The resulting crystals of Sp1 and variants, as well as all compounds, may be mounted on a Kappa goniometer and flash frozen to 100°K using the Cobra cooling device. The crystals may then be subject to CuKa X-rays and data collected using CMOS detectors. Any crystals with reasonable quality diffraction may then be stored in liquid nitrogen dewars and data collected at various synchrotrons such as BNL. The diffracted data may be used to solve the structure using standard structure determination software suites like CCP4 and PHENIX.

[00125] Cellular Thermal Shift Assays (CETSA)

[00126] CETSA may be conducted to examine target engagement of TA and lead analogs on SP1. This assay may confirm or deny direct binding of TA/analogs to SP1 , thus, confirming our theory of drug-protein interaction and validating our data. CETSA relies on the theory that unbound proteins are less stable than bound proteins. Thus, unbound proteins may unfold or ‘melt’ when exposed to a heat gradient faster than bound proteins. This difference in melting temperature is referred to as a Tm shift. The final step is to quantify the remaining levels of stabilized protein following the irreversible aggregation of thermally unfolded proteins. Cell may be prepared and exposed to analogs as stated above. CETSA samples may be separated by SDS-PAGE and immunoblotting may be performed using rabbit anti-SP1 monoclonal antibody at 4 °C. Band intensity of immunoblot films may be quantified using Imaged software (version 1.15t). Analysis of melting shift and isothermal dose-response fingerprint may be analyzed using GraphPad Prism 7 (version 7.02, Graph Pad Software) using the Boltzmann sigmoid equation and the four-parameter logistic curve. [00127] Also, expect to observe a thermal shift when SP1 and DNA binds and the same shift may be disrupted when TA or an analog is added. In the absence of Sp1 , AD- related genes such as tau are not impacted. Issues may arise if the analogs are not as effective as TA or if there is discordance between lowering tau and the impact on Sp1 DNA binding and tau promoter activity. If these outcomes present themselves, we may revise our hypothesis and see alternative approaches.

[00128] Example 3 - Identify global transcriptomic and proteomic signatures of TA and analogs.

[00129] Sp1 is a transcription factor with the ability to activate a variety of genes with GC box elements. Repression of the expression of these neurodegeneration-related genes would contribute positively to the amelioration of the pathogenesis associated with AD and other tauopathies. Therefore, it is important to know what other genes are activated or repressed in the presence of TA and its analogs. The scope of these changes may determine the potential “on or off-target” beneficial and/or detrimental effects that may occur. Furthermore, the scope of SP1 -driven gene expression needs to be defined. Transcriptom ics and proteomic analysis from cell cultures exposed to TA and analogs may be analyzed and clustered to identify the most relevant genes altered following treatment.

[00130] Cell culture and exposed tissue

[00131] Cells may be grown as above.

[00132] Proteomics

[00133] Olink biomarker discovery technology is a high throughput proteomics analysis that offers 13 target panels comprising over 1200 biomarkers. Olink’s protein biomarker panels are based on innovative immunoassay technology with several unique features such as: high multiplexing grade (92 proteins per assay and per panel), using a minimal volume of biological material (1 pl per 92 proteins), and over 1200 uniquely selected proteins representing most biological pathways. The Olink reagents are based on the Proximity Extension Assay (PEA) technology, where 92 oligonucleotide labeled antibody probe pairs bind to their respective target protein present in the sample. A PCR reporter sequence is formed by a proximity dependent DNA polymerization, amplified, and subsequently detected and quantified using real-time PCR. Olink offers panels of biomarkers that might be valuable for our study; including Neurology panel, NeuroExploratory panel, Cell regulation panel, Devlopment panel, Organ damage, Immune response panel and Imflammation panel. In the proposed study, we may screen iPSCs treated with TA, TA analogs and controls using Olink biomarkers discovery panels, and identified biomarkers from the pilot study may serve as candidate biomarkers for the development of a custom signature panel, which may be then validated in a larger study and different models.

[00134] Transcriptomics

[00135] Total RNA with a RIN number above 8 may be used as input for the sequencing library construction using the TruSeq Stranded mRNA kit (Cat #: 20020594) from Illumina following the manufacturer’s protocol. Briefly, from 500ng of total RNA, mRNA molecules may be captured by using poly-T oligo attached magnetic beads and then mRNA may be fragmented for the required size. cDNA may be generated from the cleaved RNA fragments using random priming during first and second strand synthesis. Barcoded DNA adapters may be ligated to both ends of the DNA and then amplified by PCR. The quality of the library generated may be checked on an Agilent 2100 Bioanalyzer system and quantified using the Qubit system. Libraries that pass quality control may be pooled, clustered on a cBot platform, and sequenced on an Illumina HiSeq 4000 at a minimum of 20 million paired end reads (2x75 bp) per sample.

[00136] Gene expression data processing and analysis

[00137] Our primary interest is to identify genes with transcription response that is altered by TA and its analogs. For each gene, we may test the null hypothesis that the mean expression change associated with treatment is the same between cells exposed to TA and unexposed controls. [00138] To account for the large number of simultaneous comparisons and to control false discoveries, we may use Empirical Bayes methods such as Significance Analysis of Microarrays (SAM) or Linear model for microarray data (limma) to perform moderated hypothesis tests. Functional analysis, including Geneontology analysis and Gene Set Enrichment Analysis may be performed to identify the biological pathways involved in the gene expression response.

[00139] Expected Outcomes

[00140] Tissue microarray studies following exposure to environmental agents that disrupt gene expression via SP1 demonstrate that less than 1 % of genes are affected (unpublished results). Studies mentioned above in cancer cell lines agree with our findings. Furthermore, the majority of genes that do possess such poly-G regions are housekeeping genes, featuring little, if any, regulation and minimal transcriptional activity. Therefore, we do not expect that TA or the analogs would result in any widespread disturbances of gene expression; however, we may, also, identify any relevant target gene whose expression is altered by Sp1 and determine their role as to any potential toxicity. We expect to find repression of genes that are driven by Sp1 . This would confirm that their suppression may be related to the lowering of Sp levels in these cells caused by TA. We expect that other Sp driven genes to be lowered as well. Housekeeping genes, which are resistant to these effects, may be spared as well as genes which lack GC or CG elements in their promoters. We expect to find characteristic patterns of expression associated with TA exposure.

[00141] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[00142] The terms “a,” “an,” “the” and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[00143] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00144] Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[00145] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.

[00146] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[00147] In closing, it is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

Claims

What is claimed is:

1 . A pharmaceutical composition comprising a compound of Formula I or Formula II,

Formula I Formula II or a pharmaceutically acceptable carrier, wherein Y is S, NH, 0 or CH2;

R¹ is H or C1-6 alky; and

R², R³, R⁴, R⁵, R⁶, and Z are each independently H, F, Cl, Br, NO2, CF3, CN, C1-6 alky, or

OCnH2n+i, wherein n is 1 to 6.

2. The pharmaceutical composition of claim 1 , wherein the composition comprises the compound of Formula I, and wherein Y is S; R¹, R³, R⁴, R⁵, R⁶ and Z are H; and R² is CN.

3. The pharmaceutical composition of claim 1 , wherein the composition comprises the compound of Formula I, and wherein Y is S; R¹, R² and Z are H; and one of R³, R⁴, R⁵, or R⁶ is CN.

4. The pharmaceutical composition of claim 1 , wherein the composition comprises the compound of Formula II, and wherein Y is NH; R¹, R², R³, R⁴, R⁶ and Zare H; and R⁵ is CF3.

5. The pharmaceutical composition of claim 1 , wherein the composition comprises the compound of Formula II, and wherein Y is NH; R¹, R⁵ and Z are H; and one of R², R³, R⁴, or R⁶ is CF₃.

6. The pharmaceutical composition of claim 1 , wherein the compound of Formula I or the compound of Formula II comprises

or a pharmaceutically acceptable salt thereof.

7. A method of down-regulating tau protein expression or activity in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I or Formula II,

or a pharmaceutically acceptable carrier, wherein Y is S, NH, 0 or CH2;

R¹ is H or C1-6 alky; and R², R³, R⁴, R⁵, R⁶, and Z are each independently H, F, Cl, Br, NO2, CFs, CN, C1-6 alky, or OCnH2n+i, wherein n is 1 to 6.

8. The method of claim 7, wherein the composition comprises the compound of Formula I, and wherein Y is S; R¹, R³, R⁴, R⁵, R⁶ and Zare H; and R² is CN.

9. The method of claim 7, wherein the composition comprises the compound of Formula I, and wherein Y is S; R¹, R² and Z are H; and one of R³, R⁴, R⁵, or R⁶ is CN.

10. The method of claim 7, wherein the composition comprises the compound of Formula II, and wherein Y is NH; R¹, R², R³, R⁴, R⁶ and Z are H; and R⁵ is CFs.

11. The method of claim 7, wherein the composition comprises the compound of Formula I, and wherein Y is NH; R¹, R⁵ and Z are H; and one of R², R³, R⁴, or R⁶ is CFs.

12. The method of claim 7, wherein the compound of Formula I or the compound of Formula II comprises

13. A method of of treating a neurodegenerative disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound of Formula I or Formula II,

Formula I Formula II or a pharmaceutically acceptable carrier, wherein Y is S, NH, 0 or CH2;

R¹ is H or C1-6 alky; and

R², R³, R⁴, R⁵, R⁶, and Z are each independently H, F, Cl, Br, NO2, CF3, CN, C1-6 alky, or

OCnH2n+i, wherein n is 1 to 6.

14. The method of claim 13, wherein the composition comprises the compound of Formula I, and wherein Y is S; R¹, R³, R⁴, R⁵, R⁶ and Zare H; and R² is CN.

15. The method of claim 13, wherein the composition comprises the compound of Formula I, and wherein Y is S; R¹, R² and Z are H; and one of R³, R⁴, R⁵, or R⁶ is CN.

16. The method of claim 13, wherein the composition comprises the compound of Formula II, and wherein Y is NH; R¹, R², R³, R⁴, R⁶ and Z are H; and R⁵ is CF3.

17. The method of claim 13, wherein the composition comprises the compound of Formula I, and wherein Y is NH; R¹, R⁵ and Z are H; and one of R², R³, R⁴, or R⁶ is CF3.

18. The method of claim 13, wherein the compound of Formula I or the compound of Formula II comprises

19. The method of claim 13, wherein said neurodegenerative disease comprises a tauopathy.

20. The method of claim 13, wherein said neurodegenerative disease comprises Alzheimer’s Disease.