Amyloid beta oligomerization inhibitors and their therapeutic uses

Abstract

Disclosed are compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds and pharmaceutical compositions for treating and / or preventing diseases or disorders associated with the biological activity of amyloid beta in a subject in need thereof. The disclosed compounds include cyclohexane 1,3-dione, which can inhibit one or more biological activities of amyloid beta, such as amyloid beta oligomerization. Thus, the disclosed compounds and pharmaceutical compositions can be utilized in methods of treating and / or preventing diseases or disorders associated with amyloid beta activity in a subject in need thereof, which diseases and disorders can be associated with amyloid beta oligomerization.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 63 / 203,245, filed July 14, 2021, the entire contents of which are incorporated by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grants AG061708 and AG050492 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (702581.02174.xml; size: 5,531 bytes; and creation date: July 13, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0004] The field of the invention relates to small molecule inhibitors of amyloid beta (Aβ) oligomerization and their use in treating diseases and disorders associated with Aβ oligomerization.

[0005] Alzheimer's disease currently afflicts 5.8 million Americans. It is the sixth leading cause of death in the United States. Amyloid-β peptide oligomers and hyperphosphorylated tau are deeply involved in memory loss in Alzheimer's. Amyloid-β oligomers are observed in the brains of Alzheimer's disease patients. These Amyloid-β oligomers bind to neurons and induce tau phosphorylation and neuronal damage.

[0006] There are currently no FDA approved drugs for the treatment of Alzheimer's disease that slow or halt disease progression, and none that directly act on toxic oligomers, therefore there is a need in the field for therapeutics that target pathogenic amyloid-β oligomers. Summary of the Invention

[0007] Disclosed herein is the use of (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione or a pharma- ceutically acceptable salt thereof in methods for treating, modulating, and detecting candidate compounds for amyloid-β oligomerization. Also disclosed is a pharmaceutical composition or unit dose package comprising (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione, or a pharma- ceutically acceptable sale thereof. (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione has the following structural formula:

[0008] [ka]

[0009] and can also be called NU-9.

[0010] In one embodiment of the disclosure, a method of treating Alzheimer's disease in a subject in need thereof is provided. In some embodiments, the method comprises administering (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione, or a suitable pharmaceutical salt thereof, to the subject to treat Alzheimer's disease in the subject. In some embodiments, the method treats memory loss in the subject. In some embodiments, the subject suffers from amyloid-β oligomerization. In some embodiments, the subject is administered a daily dosage of about 100 mg / kg, 75 mg / kg, 50 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 5 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.01 mg / kg or less of the compound, or within a range limited to any of these values. In some embodiments, the compound is administered orally. In some embodiments, the method further comprises administering a cholinesterase inhibitor and / or an N-methyl-D-aspartate receptor antagonist. In some embodiments, the method comprises administering a cholinesterase inhibitor selected from the group consisting of galantamine, rivastigmine, and donepezil. In some embodiments, the method comprises administering the N-methyl-D-aspartate receptor antagonist memantine.

[0011] In another aspect of the disclosure, a method for treating or preventing a disease or disorder associated with amyloid-β oligomerization in a subject in need thereof is provided. In some embodiments, the method comprises administering (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione, or a suitable pharmaceutical salt thereof, to the subject to treat a disease or disorder associated with amyloid-β oligomerization in the subject. In some embodiments, the disease or disorder is selected from Alzheimer's disease, cerebral amyloid angiopathy (CAA), inflammatory cerebral amyloid angiopathy, frontotemporal dementia, and cerebral amyloidoma. In some embodiments, the method treats memory loss in the subject. In some embodiments, the subject is administered a daily dose of about 100 mg / kg, 75 mg / kg, 50 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 5 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.01 mg / kg or less of the compound, or within a range limited to any of these values. In some embodiments, the compound is administered orally. In some embodiments, the method further comprises administering a cholinesterase inhibitor and / or an N-methyl-D-aspartate receptor antagonist. In some embodiments, the method comprises administering a cholinesterase inhibitor selected from the cholinesterase inhibitors galantamine, rivastigmine, and donepezil. In some embodiments, the method comprises administering the N-methyl-D-aspartate receptor antagonist memantine.

[0012] In another aspect of the present disclosure, a method for modulating amyloid-β oligomerization activity in the brain of a subject is provided. In some embodiments, the method comprises administering (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione, or a suitable pharmaceutical salt thereof, to the subject to modulate amyloid-β oligomerization activity in the brain of the subject. In some embodiments, the method inhibits the formation of neuron-bound amyloid-β oligomers. In some embodiments, the method promotes the formation of unbound amyloid-β oligomers. In some embodiments, the subject suffers from Alzheimer's disease. In some embodiments, the method treats memory loss in the subject. In some embodiments, the subject suffers from amyloid-β oligomerization in the brain. In some embodiments, the subject is administered a daily dose of about 100 mg / kg, 75 mg / kg, 50 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 5 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.01 mg / kg or less of the compound, or within a range limited to any of these values. In some embodiments, the compound is administered orally. In some embodiments, the method further comprises administering a cholinesterase inhibitor and / or an N-methyl-D-aspartate receptor antagonist. In some embodiments, the method comprises administering a cholinesterase inhibitor selected from the cholinesterase inhibitors galantamine, rivastigmine, and donepezil. In some embodiments, the method comprises administering the N-methyl-D-aspartate receptor antagonist memantine.

[0013] In another aspect of the present disclosure, a method for detecting a candidate compound that modulates amyloid-β oligomerization in the presence of cells is provided. In some embodiments, the method includes the steps of (i) culturing cells with amyloid-β peptide in the presence and absence of a candidate compound and contacting the control cells with amyloid-β peptide in the presence and absence of a control compound; (ii) detecting one or more parameters associated with amyloid-β oligomerization in the cells of step (i); (iii) calculating the change in one or more parameters between the cells cultured in the presence and absence of the candidate compound and calculating the change in one or more parameters between the cells cultured in the presence and absence of the control compound to generate a control index, where the control compound is (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione or a pharma- ceutically acceptable salt thereof, and the candidate compound modulates amyloid-β oligomerization if the value of the test index is equal to or improved over the value of the control index. In some embodiments, the cell is a neuron or is derived from a neuron. In some embodiments, the cells are E18 hippocampal neurons. In some embodiments, the one or more parameters include detecting amyloid-β oligomers (AβOs) bound to the cells.

[0014] In another aspect of the present disclosure, a unit dose package is provided. In some embodiments, the unit dose package comprises: (i) (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione, or a pharma- ceutically acceptable salt thereof; and (ii) a cholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist. In some embodiments, the cholinesterase inhibitor is selected from galantamine, rivastigmine, and donepezil. In some embodiments, the N-methyl-D-aspartate receptor antagonist is memantine.

[0015] In another embodiment of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises (i) (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione, or a pharma- ceutically acceptable salt thereof; (ii) a cholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist; and (iii) a pharma- ceutically acceptable carrier or excipient. In some embodiments, the cholinesterase inhibitor is selected from galantamine, rivastigmine, and donepezil. In some embodiments, the N-methyl-D-aspartate receptor antagonist is memantine. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows the effect of NU-9 on the binding of oligomers to neurons. Neurons were pretreated with 3 μM NU-9 for 30 min and then treated with Aβ42 monomers for 24 h. Cells were fixed with 3.7% formaldehyde, stained for AβOs (NU4, green) and tau phosphorylated at Ser396 (pSer396, red) and mounted on slides before imaging. Analysis showed that the presence of NU-9 significantly reduced the number of AβOs bound to dendrites (p=0.001, 5 samples). [Diagram 2] Figure 2 shows neurons pretreated with 3 μM NU-9 for 30 min followed by treatment with Aβ42 monomer for 26 h. Cells were fixed with 3.7% formaldehyde, stained for AβOs (NU4, green), tau phosphorylated at Thr205 (pThr205, red), and nuclei (DAPI, blue) and mounted on slides before imaging. Analysis showed that the presence of NU-9 significantly reduced the number of AβOs bound to dendrites (p=0.0005, 13-15 samples). [Diagram 3] Neurons were pretreated with 3 μM NU-9 for 30 min and then treated with Aβ42 monomer for 30 min. Cells were fixed with 3.7% formaldehyde, stained for AβOs (NU2, green), β-III tubulin (TUBB3, red), and nuclei (DAPI, blue), and mounted on slides before imaging. [Figure 4] E18 hippocampal neurons were cultured for 21 div and pretreated with 3, 15, or 30 μM NU-9 for 30 min, followed by treatment with 500 nM Aβ42 for 30 min. Cells were fixed with 3.7% formaldehyde, stained for AβOs (NU2, green), β-III tubulin (red), and nuclei (DAPI, blue), and mounted on slides before imaging at 63x. More extensive inhibition of AβOs was observed with higher concentrations of NU-9. [Figure 5A] A) NU-9 and Aβ were mixed in F12 medium in the absence of cells and incubated at 37°C. [Figure 5B] B) AβO concentrations were probed with NU2 antibody by dot blot after 24 hours in the presence or absence of Aβ and 3 μM NU-9. No significant differences were observed between solutions containing 3 μM NU-9 and Aβ and solutions containing Aβ and vehicle. [Figure 6] Dishes without cells were fixed with 3.7% formaldehyde for 30 min under conditions mimicking cell culture, with 3 μM NU-9 and 500 nM Aβ42, stained for AβOs (NU2, green), and mounted on slides before imaging at 63x. The presence of NU-9 did not significantly affect the number of AβOs bound to the coverslip (p=0.5). [Figure 7] NU-9 attenuates memory loss in 5xFAD old mice. Preliminary results obtained in 11-month-old mice treated with NU-9 for 4 weeks. Memory performance was assessed before and after treatment with vehicle or NU-9. All animals showed normal motor and exploratory behavior; not shown. [Figure 8] NU-9 is effective even when washed out before adding Aβ monomer. Mature hippocampal neurons were treated with DMSO control (control) or NU-9. NU-9 or vehicle was then washed out and Aβ was added (500 nM, 30 min). Cells were labeled with NU2 (green, anti-AβO) and MAP2 (red, anti-dendritic). [Figure 9]The effect of NU-9 requires the presence of cells. Conditioned medium was collected from mature hippocampal neurons. DMSO control (control), Aβ monomer (Aβ), or NU-9 were added, followed by Aβ (NU-9 + Aβ in conditioned medium), and the concentration of AβOs was analyzed by dot blot. Conditioned medium was also collected from neurons treated with NU-9, to which Aβ was added (medium from NU-9 in cells + amyloid β). Aβ was not reduced in any of the treatment conditions. A standard curve was generated in medium using 0-1000 fmol AβOs. Dot blots were labeled with NU2 (anti-AβOs). [Figure 10-1] NU-9 does not reduce AβO formation from Aβ monomers in cell-free solution, nor in conditioned medium obtained from cells treated with NU-9. (Conditioned medium) Dot blots probed with NU2 were used to measure AβO abundance in solutions of DMSO vehicle (control), Aβ, and conditioned medium to which NU-9 was added followed by Aβ for 30 min each. The lower row corresponds to samples in which cells were treated with NU-9 for 30 min, then the extracellular medium was removed and Aβ was added to the medium for 30 min. The upper standard curve was generated from 0–1000 fmol of preformed AβOs. [Figure 10-2] (Fresh medium) Dot blots probed with NU2 were used to measure AβO abundance in DMSO vehicle (control), Aβ, and culture medium solutions containing NU-9 (3–300 μM) followed by Aβ for 30 min each. Each of these solutions was prepared in triplicate. The upper standard curve was generated from preformed AβOs ranging from 0 to 1000 fmol. [Figure 11] NU-9 does not reduce the binding of preformed AβOs to neurons. Hippocampal neurons were treated with 3 μM NU-9 or vehicle for 30 min, then treated with Aβ monomers or preformed AβOs for 24 h. NU-9 was able to reduce AβO binding in neurons treated with Aβ monomers, but not in neurons treated with preformed AβOs. Neurons were stained with NU4 (anti-AβO antibody) and the number of points per treatment was quantified. [Figure 12-1]NU-9 does not reduce total Aβ species. Mature hippocampal neurons were treated with NU-9 (3 μM, 30 min) followed by Aβ monomer (500 nM, 30 min). They were labeled with 6E10 (anti-total Aβ, green) and MAP2 (anti-dendritic). [Figure 12-2] The total number of Aβ particles per image was quantified as well as the fluorescence intensity of the Aβ signal (IntDen). [Figure 13] Lysosomal inhibition inhibits the action of NU-9 on synaptically bound AβOs. Mature hippocampal neurons were treated with 100 nM bafilomycin A for 30 min, then 3 μM NU-9 for 30 min, and finally 500 nM Aβ for 30 min. Cell cultures were fixed and labeled with NU2 (anti-AβO antibody) and MAP2 (anti-dendrite antibody). The number of AβOs bound per micron along the dendrite was quantified using SynPAnal. [Figure 14] Lysosomal acidification as a potential mechanism of NU-9 effect on synaptically bound AβOs. Potential actions of NU-9 are shown in orange. NU-9 may activate endosomal uptake of Aβ monomers and endolysosomal trafficking of Aβ monomers and small oligomers. Alternatively, it may activate lysosomal acidification and release of unbound oligomers, decreasing the escape of large oligomers from less acidic endolysosomal vesicles. [Figure 15-1] Mature hippocampal neurons were pretreated with vehicle or 3 μM NU-9 for 30 min, followed by vehicle, 200 nM, or 500 nM AβO for 30 min. Cells were then fixed and labeled with anti-MAP2 (green, dendrites), DAPI (blue, nuclei), and NU2 (red, AβO). At any concentration, NU-9 did not affect AβO binding to dendrites. [Figure 15-2] The graph shows preliminary quantification points / μm dendrite for vehicle, 200 nM AβO, and 200 nM AβO+9 (analysis of 10 images / condition, *** indicates p<0.001). [Figure 16-1]Lysosomal inhibition prevents the action of NU-9. Mature hippocampal neurons were pretreated with vehicle or 100 nM bafilomycin A for 30 min, then treated with vehicle or 3 μM NU-9 for an additional 30 min, followed by a final 30 min with vehicle or 500 nM Aβ monomer. [Figure 16-2] Cells were then fixed and labeled with anti-MAP2 (green, dendrites), DAPI (blue, nuclei), and NU2 (red, AβOs). As before, bafilomycin A significantly inhibited the effect of NU-9. The graph shows quantification of NU2 puncta / µm along dendrites (analysis of 15 images / condition, *** indicates p<0.001, ** indicates p<0.01). [Figure 17-1] Cysteine ​​cathepsin inhibition does not block the effects of NU-9. Mature hippocampal neurons were first treated with 10 μM E64 or vehicle for 24 h, then with 3 μM NU-9 for 30 min, and finally with 500 nM Aβ monomer for 30 min. [Figure 17-2] Cells were then fixed and labeled with anti-MAP2 (red, dendrites), DAPI (blue, nuclei), and NU2 (green, AβOs). The graph shows the quantification of dendrites in puncta / μm (20 images analyzed / condition, *** indicates p<0.001, ** indicates p<0.01). [Figure 18] Mature hippocampal neurons were treated with vehicle or 100 nM bafilomycin A for 30 min and then labeled with Lysotracker DND-99 (red) at 75 nM for 30 min or 50 nM for 2 h as indicated. Both conditions yielded clear labeling of small structures. Treatment with 75 nM for 30 min yielded a brighter signal. [Figure 19] Mature hippocampal neurons were first treated with 3 μM 9 for 30 min, then treated with A) 30 or B) 200 nM LC AβO for 30 min. Cells were then fixed and labeled with anti-MAP2, DAPI, and NU2 (AβO). Graph shows quantification of dendrites in puncta / μm (30 images analyzed / condition, ***p<0.001, **p<0.01, *p<0.05). [Figure 20]A) Mature cultures of E18 hippocampal neurons were pretreated with 3 μM NU-9 for 30 min followed by addition of 500 nM Aβ for 30 min. B) Graph shows quantification of puncta / μm dendrites (0 image analyses / condition). [Figure 21] Addition of monomeric Aβ1-42 (500 nM) to mature E18 hippocampal neurons for 30 min produced unbound extracellular AβOs detected by dot blot analysis. Pretreatment with 3 μM NU-9 for 30 min did not significantly reduce the number of unbound extracellular AβOs bound to dendrites in either the supernatant (p=0.6) or uncentrifuged solution (p=0.3). Three biological replicates were performed for each condition, each spotted in duplicate on the dot blot. [Figure 22] A) Representative images of AβO (green, NU2) bound to dendrites (red, MAP2) of mature hippocampal neurons challenged with 500 nM Aβ42 and pretreated with 3 μM NU-9 for 30 min or with or without 100 nM MG132 for 30 min, and B) Quantification of AβO puncta per μm along dendrites. These were performed using ImageJ. n=30 images / condition. [Figure 23] A) Representative images of AβO (green, NU2) bound to dendrites (red, MAP2) of mature hippocampal neurons. Mature hippocampal neurons were challenged with 500 nM Aβ42 with or without 30 min pretreatment with 3 μM NU-9 and 1 h pretreatment with 1 μM vaquolin-1. B) Quantification of AβO puncta per μm along dendrites, performed using ImageJ. n=30 images / condition. [Figure 24-1] Mature E18 hippocampal neurons were treated with 3 μM NU-9 and subsequently labeled with Lysotracker. A) Total immunofluorescence (integrated density), B) Mean intensity, C) Mean size of acidic compartments, D) Number of acidic compartments. [Figure 24-2] These values ​​were calculated using ImageJ from 10 images across two coverslips of neurons, and similar trends were observed for separate replicates in different cell cultures. [Diagram 25]A) Mature E18 hippocampal neurons were first treated with 10 μM CA-074 or vehicle for 24 h, then with 3 μM for 9 min, and finally with 500 nM Aβ monomer for 30 min. Cells were then fixed and labeled with anti-MAP2 (green, dendrites), DAPI (blue, nuclei), and NU2 (red, AβO). B) Graph shows quantification of puncta / μm dendrites (30 images analyzed / condition, ***p<0.001, **p<0.01, *p<0.05). [Figure 26-1] A) 0.0003-150 μM NU-9 was combined with purified cathepsin L enzyme in the presence of the cathepsin L substrate Z-FR-AMC. Cathepsin L enzyme activity was measured by the rate of 7-AMC production by fluorescence. The rate of change was quantified graphically. The standard inhibitor Z-FY-CHO reduced cathepsin L activity over the same concentration range (not shown). [Figure 26-2] B) Mature hippocampal neurons were treated with NU-9 for 30 min and then intracellular cathepsin L activity was monitored using Magic Red. Cells were treated in triplicate wells and 9–22 images were analyzed per condition. [Figure 27] Inhibition of cathepsin L suppresses AβO accumulation similar to the action of NU-9. Neurons were first pretreated with 10 μM cathepsin L inhibitor (Cayman) for 1 h, then with 3 μM NU-9 for 30 min, and finally with 500 nM Aβ for 30 min, then cells were fixed and labeled with anti-MAP2 (green, dendrites), DAPI (blue, nuclei), and NU2 (red, AβOs). Graph shows quantification of dendrites in puncta / μm. [Figure 28-1] 0.0003-30 μM NU-9 was combined with purified cathepsin B enzyme in the presence of the cathepsin B substrate Z-RR-AMC. Cathepsin B enzyme activity was measured by the rate of 7-AMC production by fluorescence. The percent change in rate with NU-9 was quantified graphically. The standard cathepsin B inhibitor CA-074 reduced cathepsin L activity over the same concentration range (not shown). [Figure 28-2] B) Mature hippocampal neurons were treated with NU-9 for 30 min, then intracellular cathepsin L activity was monitored using Magic Red. Cells were treated in triplicate. [Figure 29-1] Calpain inhibition may mimic the effects of NU-9. E18 hippocampal neurons at 21 div were treated with 10 μM MDL-28170 for 30 min, then 3 μM NU-9 for 30 min, and finally 500 nM Aβ for 30 min. Cells were labeled with AβOs, NU2, and MAP2 by immunofluorescence for neuronal dendrites. [Figure 29-2] The number of AβOs per micron along the dendrite was quantified using ImageJ for 30 images for each condition and the data were analyzed using Prism. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention is described herein using several definitions, as set forth below and throughout the application.

[0018] definition The disclosed subject matter can be further described using the following definitions and terminology: The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0019] As used in the specification and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, the term "substituent" should be interpreted to mean "one or more substituents" unless the context clearly dictates otherwise.

[0020] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of skill in the art and will vary to some extent depending on the context in which they are used. If there are uses of the terms that are not clear to a person of ordinary skill in the art given the context in which the terms are used, "about" and "approximately" will mean up to ±10% of the particular term, and "substantially" and "significantly" will mean greater than or exceeding ±10% of the particular term.

[0021] As used herein, the terms "include" and "including" should be construed as "open" transitional terms that allow for the inclusion of additional elements beyond those recited in the claims. The terms "consist" and "consisting of" should be construed as "closed" transitional terms that do not allow for the inclusion of additional elements beyond those recited in the claims. The term "consisting essentially of" should be construed as partially closed and can only include additional elements that do not fundamentally change the nature of the claimed subject matter.

[0022] The phrase "such" should be interpreted as "including, for example," and further, the use of any and all exemplary language, including but not limited to "such," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0023] Additionally, when a convention similar to "at least one of A, B, C, etc." is used, such configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "A system having at least one of A, B, C includes, but is not limited to, a system having only A, only B, only C, between A and B, between A and C, between B and C, and / or at least one of A, B, and C"). It will be further understood by one of ordinary skill in the art that virtually any disjunction and / or phrase presenting two or more alternative terms, either in the specification or in the drawings, should be understood to consider the possibility of including either of the terms, or either of both terms, or one of either terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0024] All language such as "up to," "at least," "greater than," "less than," etc., refers to ranges that are inclusive of the recited numbers and can subsequently be broken down into ranges and subranges. Ranges include each individual member. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, etc.

[0025] The modal verb "may" refers to a preferred use or selection of one or more options or choices of several described embodiments or features included within the same. When no options or choices are disclosed regarding a particular embodiment or feature included within the same, the modal verb can refer to a positive action regarding the manner and aspects of making or using the described embodiment or feature included within the same, or a definitive decision to use a particular skill regarding the described embodiment or feature included within the same. In this latter context, the modal verb has and can have the same meaning and meaning as the modal verb "can."

[0026] As used herein, a "subject in need thereof" can refer to a subject in need of treatment for a disease or disorder associated with amyloid-β activity and / or expression. A subject in need thereof can include a subject having a disease or disorder characterized by amyloid-β oligomerization or aggregation. As used herein, a "subject in need thereof" includes, but is not limited to, a subject in need of treatment for Alzheimer's disease, cerebral amyloid angiopathy, inflammatory cerebral amyloid angiopathy, and cerebral amyloidoma.

[0027] The term "subject" may be used interchangeably with the terms "individual" and "patient," and includes human and non-human mammalian subjects.

[0028] The disclosed compounds, pharmaceutical compositions, and methods can be utilized to treat and / or prevent diseases and disorders associated with amyloid-β oligomerization, which may include, but are not limited to, Alzheimer's disease, cerebral amyloid angiopathy (CAA), inflammatory cerebral amyloid angiopathy, and cerebral amyloidoma.

[0029] The disclosed compounds can be utilized to modulate the biological activity of amyloid-β, including modulating the oligomerization activity of amyloid-β.

[0030] Amyloid-β refers to the protein derived from cleavage of the amyloid-β precursor protein (APP) by the enzymes β-secretase and γ-secretase. The amyloid-β peptide can be 36-43 amino acids in length.

[0031] Amyloid-β is produced, for example, by proteolytic processing of the amyloid precursor protein (APP) by α- and γ-secretases. The following amino acid sequence is an example of the sequence of APP (SEQ ID NO:1):

[0032] [ka]

[0033] Thus, it will be apparent to those skilled in the art that several possible isoforms of amyloid beta generated by proteolytic processing from APP may be found in a subject in need of treatment and / or prevention of a disease or disorder associated with amyloid beta oligomerization. Below are the amino acid sequences for non-limiting examples of amyloid beta isoforms:

[0034] Amyloid beta 1-40 (aa 672-711 of APP) has the following amino acid sequence (SEQ ID NO:2):

[0035] [ka]

[0036] Amyloid beta 1-42 (aa 672-713 of APP) has the following amino acid sequence (SEQ ID NO:3):

[0037] [ka]

[0038] The amyloid beta may include an N-terminal truncation, for example an N-terminal truncation of the first two amino acid residues, referred to as N3 amyloid beta, such as amyloid beta (3-40) or amyloid beta (3-42). The amyloid beta may include a pyroglutamic acid residue (pE) replacing a glutamic acid residue. In some embodiments, the amyloid beta includes an N-terminal truncation of the first two amino acids and a pyroglutamic acid residue (pE) at the third position (N3pE), where it is the first position. Such an amyloid beta may have the sequence set forth in (SEQ ID NO: 4).

[0039] [ka]

[0040] Various isoforms of amyloid-β (Ab) differ in their aggregation properties. The amyloid-β pool is characterized by three major groups: monomers, soluble oligomers, and insoluble fibrils. Each pool can encompass multiple structures based on different organizations. The term "amyloid-β oligomerization" refers to the formation of soluble oligomers. Such soluble oligomers can be organized into different structures consisting of two or more Aβ, such as dimers, trimers, tetramers, pentamers, decamers, and dodecamers. Soluble oligomers can also include, for example, Aβ-derived diffusible ligands (ADDLs) and Aβ*56.

[0041] Toxic soluble oligomers, distinct from fibril-like monomers or higher aggregates, have been identified in AD brains. There is an inverse correlation between the size of Aβ aggregates and the strength of their toxicity. As the size of oligomeric aggregates increases, their deleterious effects decrease. Aβ dimers have been shown to form more stable high molecular weight structures called protofibrils that are neurotoxic. Thus, the dimeric unit of Aβ has been considered important as a component of toxic aggregates. Thus, oligomerization of Aβ involves the formation of toxic amyloid beta oligomers (AβOs).

[0042] AβOs are harmful to neurons and may be a causative agent of neurodegeneration observed, for example, in Alzheimer's disease. Cultured cells, such as cultured neurons, and cells in contact with antibodies produce AβOs bound to the cell surface. However, we have found that the compound NU-9 modulates antibody oligomerization activity.

[0043] As used herein, with respect to Ab oligomerization activity, "modulate" refers to the effect on Ab aggregation. Modification of an antibody can refer to modulation of the Aβ pool (e.g., the relative proportions between Ab monomers, soluble oligomers, and insoluble fibrils), modification of soluble oligomers (e.g., the relative proportions between dimers, trimers, tetramers, pentamers, decamers, etc.), modulation of the organization of soluble oligomers, or any combination thereof.

[0044] As shown in the Examples, NU-9 effectively reduced the number of pathological species of AβOs generated from antibodies capable of binding to the surface of cells (Figure 2). NU-9 also promoted the formation of unbound oligomers. The inventors also discovered that NU-9 acts via a lysosome-dependent mechanism to reduce the formation of toxic AβOs (Figures 25-29). Thus, in some embodiments, the method includes inhibiting the formation of neuron-bound AβOs or toxic AβOs.

[0045] How to use In one embodiment of the present disclosure, a method for treating Alzheimer's disease in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject an effective amount of NU-9 or a suitable pharmaceutical salt thereof to treat Alzheimer's disease in the subject.

[0046] In another aspect of the present disclosure, a method of treating or preventing a disease or disorder associated with amyloid-β oligomerization in a subject in need thereof is provided.

[0047] In another embodiment of the present disclosure, a method for regulating amyloid-β oligomerization activity in the brain of a subject is provided.In some embodiments, the method comprises administering to the subject an effective amount of NU-9 or its suitable pharmaceutical salt to regulate amyloid-β oligomerization activity in the brain of the subject.

[0048] In some embodiments, the method of the present disclosure further comprises administering to the subject at least one other compound selected from a cholinesterase inhibitor and an N-methyl-D-aspartate (NMDA) receptor antagonist. In some embodiments, the cholinesterase inhibitor is selected from galantamine, rivastigmine, and donepezil. In some embodiments, the NMDA receptor antagonist is memantine.

[0049] Disclosed are compounds, pharmaceutical compositions comprising the compounds, and methods of using the compounds and pharmaceutical compositions for treating and / or preventing diseases or disorders associated with amyloid beta biological activity in a subject in need thereof. The disclosed compounds can include cyclohexane 1,3-diones, such as NU-9 and pharma- ceutically acceptable salts thereof, that inhibit one or more biological activities of amyloid beta, such as amyloid beta oligomerization. Thus, the disclosed compounds and pharmaceutical compositions can be utilized in methods of treating a subject having or at risk of developing a disease or disorder associated with amyloid beta activity, which can be a disease or disorder associated with amyloid beta oligomerization.

[0050] The disclosed compounds include cyclohexane 1,3-diones. Cyclohexane 1,3-diones and methods for synthesizing cyclohexane 1,3-diones are disclosed in the art (see, for example, Zhang et al., "Chiral Cyclohexan 1,3-diones as Inhibitors of Mutant SOD1-Dependent Protein Aggregation for the Treatment of ALS," ACS Medic. Chem. Lett., 20021, 3, 584-587, the contents of which are incorporated herein by reference in their entirety). The disclosed compounds for the uses disclosed herein include, but are not limited to, (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione (NU-9).

[0051] In some embodiments, the disclosed methods can be performed to treat and / or prevent a disease or disorder selected from, but not limited to, Alzheimer's disease, cerebral amyloid angiopathy, inflammatory cerebral amyloid angiopathy, frontotemporal dementia, and cerebral amyloidoma. In some embodiments, the disclosed methods can be performed to treat and / or prevent one or more symptoms of a disease or disorder associated with amyloid-β activity.

[0052] The disclosed methods can be performed to treat and / or prevent memory loss in a subject. For example, the disclosed methods can be performed to treat and / or prevent memory loss in a subject associated with amyloid-β oligomerization and impaired neuronal function.

[0053] In the disclosed methods, a subject may be administered an effective amount of a disclosed compound to treat and / or prevent amyloid beta oligomerization in the subject. In some embodiments of the disclosed methods, the subject is administered a daily dose of about 100 mg / kg, 75 mg / kg, 50 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 5 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.01 mg / kg or less of the disclosed compound, or within a range limited to any of these values.

[0054] In the disclosed methods, the compounds and pharmaceutical compositions can be administered to a subject by any suitable route to deliver an effective amount of the disclosed compounds to a site in the subject exhibiting amyloid β oligomerization or to a site in the subject at risk of undergoing amyloid β oligomerization, such as the subject's brain. In some embodiments, the compounds and pharmaceutical compositions are administered via the oral route.

[0055] Pharmaceutical Compositions In another embodiment of the present disclosure, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises (i) NU-9, or a pharma- ceutically acceptable salt thereof, and (ii) a pharma- ceutically acceptable carrier or excipient.

[0056] In some embodiments, the pharmaceutical composition combines NU-9 with another compound for use in treating Alzheimer's disease. In some embodiments, the pharmaceutical composition comprises (i) NU-9, or a pharma- ceutically acceptable salt thereof; (ii) a cholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist; and (iii) a pharma- ceutically acceptable carrier or excipient. In some embodiments, the cholinesterase inhibitor is selected from galantamine, rivastigmine, and donepezil. In some embodiments, the N-methyl-D-aspartate receptor antagonist is memantine.

[0057] The compounds used in the compositions and methods disclosed herein can be administered as pharmaceutical compositions, and thus pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions can take any pharma-ceutically acceptable physical form, and illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of the disclosed compounds, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain a daily dose of a given compound, or each dosage unit may contain a fraction of the daily dose, e.g., 1 / 2 or 1 / 3. The amount of each compound to be contained in each dosage unit may depend, in part, on the identity of the particular compound selected for treatment, and other factors such as the indication for which it is given. The pharmaceutical compositions disclosed herein can be formulated to provide quick, sustained, or delayed release of the active ingredient after administration to a patient by using well-known methods.

[0058] Compounds for use in the methods disclosed herein can be administered as a single compound or a combination of compounds. For example, a compound that inhibits the biological activity of amyloid beta can be administered as a single compound or in combination with another compound that inhibits the biological activity of amyloid beta or has a different pharmacological activity.

[0059] As mentioned above, pharma- ceutically acceptable salts of compounds are contemplated and can also be utilized in the disclosed methods. As used herein, the term "pharma- ceutically acceptable salts" refers to salts of compounds that are not substantially toxic to living organisms. Exemplary pharma- ceutically acceptable salts include salts prepared by reacting a pharma- ceutical compound disclosed herein with a pharma- ceutical inorganic or organic acid, or an organic or inorganic base. Such salts are known as acid addition salts and base addition salts. It will be understood by those skilled in the art that most or all of the compounds disclosed herein can form salts, and that pharmaceutical salt forms are commonly used because they are often easier to crystallize and purify than the free acid or base.

[0060] Acids commonly used to form acid addition salts can include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharma- ceutically acceptable salts include sulfate, hydrogen sulfate, bisulfate, dihydrogen phosphate, phosphate, iodide, acetate, propionate, caprylate, formate, dihydrochloride, caproate, heptanolate, oxalate, malonate, succinate, cebalate, fumarate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenylacetate, phenylpropionate, citrate, α-hydroxybutyrate, glycolate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

[0061] Base addition salts include those derived from inorganic bases such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0062] The particular counterion forming a part of any salt of the compounds disclosed herein is not critical to the activity of the compound, so long as the overall salt is pharmacologically acceptable and the counterion does not impart undesirable properties to the overall salt, including undesirable solubility or toxicity.

[0063] Pharmaceutically acceptable esters and amides of compounds can also be used in the compositions and methods disclosed herein.Examples of suitable esters include alkyl, aryl, and arylalkyl esters, such as methyl ester, ethyl ester, propyl ester, dodecyl ester, benzyl ester, etc.Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methylethyl amide, etc.

[0064] Additionally, the methods disclosed herein can be practiced using solvated forms of the compounds or their salts, esters and / or amides, including ethanol solvates, hydrates, and the like.

[0065] The pharmaceutical composition can be used in a method for treating a disease or disorder associated with the biological activity of amyloid beta. As used herein, the term "treating" or "treat" means to alleviate symptoms, temporarily or permanently eliminate the cause of the resulting symptoms, and / or prevent or delay the appearance of the named disease or disorder, or reverse the progression or severity of the resulting symptoms, respectively. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0066] As used herein, the term "effective amount" refers to an amount or dose of a compound, upon single or multiple administration to a subject, that provides a desired effect in the subject under diagnosis or treatment. The disclosed methods can include administering an effective amount of the disclosed compounds (e.g., present in a pharmaceutical composition) to treat a disease or disorder associated with the biological activity of amyloid beta.

[0067] The effective amount can be easily determined by the attending physician, as one skilled in the art, by using known techniques and observing results obtained under analogous circumstances. In determining the effective amount or dosage of the compound to be administered, many factors can be taken into account by the attending physician, such as the species of the subject, its size, age, and general health, the degree of involvement or severity of the disease or disorder involved, the response of the individual subject, the particular compound to be administered, the method of administration, the bioavailability characteristics of the formulation to be administered, the selected method of administration, the use of concomitant drugs, and other relevant circumstances.

[0068] A typical daily dose can contain from about 0.01 mg / kg to about 100 mg / kg (e.g., from about 0.05 mg / kg to about 50 mg / kg and / or from about 0.1 mg / kg to about 25 mg / kg) of each compound used in the present methods of treatment.

[0069] The compositions can be formulated in unit dosage form, each dosage containing about 1 to about 1000 mg of each compound individually, or in a single unit dosage form, such as about 5 to about 300 mg, about 10 to about 100 mg, and / or about 25 mg. The term "unit dosage form" refers to physically discrete units suitable for administering to a patient, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect in association with a suitable pharmaceutical carrier, diluent, or excipient.

[0070] Oral administration is an exemplary route for administering the compounds used in the compositions and methods disclosed herein. Other exemplary routes of administration include transdermal, transdermal, intravenous, intramuscular, intranasal, intrabuccal, intrathecal, intracerebral, or intrarectal routes. The route of administration can be varied in any manner limited by the physical properties of the compound used and the convenience of the subject and the caregiver.

[0071] As will be understood by those skilled in the art, suitable formulations include those suitable for more than one route of administration. For example, the formulation may be suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those suitable for only one route of administration, and those suitable for one or more routes of administration but not suitable for one or more other routes of administration. For example, the formulation may be suitable for oral administration, transdermal administration, transdermal administration, intravenous administration, intramuscular administration, intranasal administration, buccal administration, and / or intrathecal administration, but not suitable for intracerebral administration.

[0072] The inactive ingredients and formulation methods of the pharmaceutical compositions are conventional. Conventional formulation methods used in pharmaceutical sciences can be used herein. All conventional types of compositions can be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdermal patches, and suspensions. In general, the compositions contain from about 0.5% to about 50% of the total compound, depending on the desired dose and the type of composition used. However, the amount of compound is best defined as an "effective amount", i.e., the amount of compound that provides the desired dose to a patient in need of such treatment. The activity of the compounds used in the compositions and methods disclosed herein is not expected to depend significantly on the nature of the composition, and thus the compositions can be selected and formulated primarily, or solely, for convenience and economy.

[0073] Capsules are prepared by mixing the compound with a suitable diluent and filling the appropriate amount of the mixture into capsules. Conventional diluents include inert powdered substances (such as starch), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), flours, and similar edible powders.

[0074] Tablets are prepared by direct compression, wet granulation, or dry granulation. Their formulations usually include diluents, binders, lubricants, and disintegrants (in addition to the compound). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (such as lactose, fructose, glucose, etc.). Natural and synthetic gums can also be used, including gum arabic, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.

[0075] Tablets can be coated with sugar, for example, as a flavor enhancer and sealant.Compounds can also be formulated as chewable tablets by using a large amount of pleasant-tasting substances, such as mannitol, in the formulation.For example, quick-dissolving tablet-like formulations can also be used to ensure that patients consume the dosage form and avoid the difficulties some patients experience when swallowing solids.

[0076] In tablet formulations, lubricants may be used to prevent the tablet and punches from sticking to the die. Lubricants may be selected from slippery solids such as talc, magnesium and calcium stearates, stearic acid and hydrogenated vegetable oils.

[0077] Tablets can also contain disintegrants. Disintegrants are substances that swell when moistened to break down the tablet and release the compound. Include starches, clays, cellulose, algins, and gums. Further examples include corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose.

[0078] The composition can be formulated as an enteric formulation, for example, to protect the active ingredient from the strong acid content of the stomach. Such a formulation can be produced by coating a solid dosage form with a film of a polymer that is insoluble in an acidic environment and soluble in a basic environment. Exemplary films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0079] The compounds can also be delivered using transdermal patches. The transdermal patch can include a resin composition in which the compound dissolves or partially dissolves; and a film that protects the composition and holds the resin composition in contact with the skin. Other more complex patch compositions can also be used, such as those with a membrane perforated with multiple holes through which the drug is pumped by osmosis.

[0080] As will be appreciated by one of skill in the art, formulations can be prepared with materials (e.g., excipients, carriers (e.g., cyclodextrins), diluents, etc.) that have properties (e.g., purity) that make the formulation suitable for administration to humans. Alternatively, formulations can be prepared using substances that have purity and / or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.

[0081] Unit Dose Package In another embodiment of the present disclosure, a unit dosage package is provided. The unit dosage package includes a first unit dosage containing a first drug, such as NU-9, or a pharma- ceutically acceptable salt thereof. The unit dosage package may also include a second unit dosage containing a second drug. The unit dosage package may include a container or label indicating the name, strength, control number, expiration date, administration instructions, or any combination thereof, for one or more drugs in the unit dosage package.

[0082] In some embodiments, the unit dose package comprises NU-9 or a pharma- ceutically acceptable salt thereof for treating amyloid-β oligomerization, for example as observed in Alzheimer's disease. In some embodiments, the unit dose package comprises (i) NU-9 or a pharma- ceutically acceptable salt thereof; and (ii) a cholinesterase inhibitor or an N-methyl-D-aspartate receptor antagonist. In some embodiments, the cholinesterase inhibitor is selected from galantamine, rivastigmine, and donepezil. In some embodiments, the N-methyl-D-aspartate receptor antagonist is memantine.

[0083] Methods for detecting candidate compounds In another aspect of the present disclosure, a method for detecting a candidate compound that modulates amyloid-β oligomerization in the presence of cells is provided. In some embodiments, the method includes (i) culturing cells with amyloid-β peptide in the presence and absence of a candidate compound and contacting control cells with amyloid-β peptide in the presence and absence of NU-9 or a pharma-ceutically acceptable salt thereof; (ii) detecting one or more parameters associated with amyloid-β oligomerization in the cells of step (i). (iii) calculating the change in one or more parameters between the cells cultured in the presence and absence of the candidate compound and calculating the change in one or more parameters between the cells cultured in the presence and absence of a control compound to generate a control index, where the candidate compound modulates amyloid-β oligomerization if the value of the test index is equal to or improved over the value of the control index.

[0084] As used herein, the use of the phrase "as improved compared to the value of a control index" refers to a test index having a value greater than the control index in situations where a greater value is associated with a beneficial effect, or a value decreased from the control index in situations where a decreased value is associated with a beneficial effect. This phrase reflects that some values ​​associated with a beneficial effect may be increased in the presence of a control compound, e.g., in the production of non-pathological AbO species, or decreased in the presence of a control compound, e.g., in the production of pathological AbO species.

[0085] In some embodiments, the one or more parameters associated with oligomerization include detecting the amount or relative amount of AbO on the surface of a cell, which can be determined through a variety of techniques known in the art, such as fluorescence microscopy, flow cytometry, or other means known in the art to label a protein and detect the protein on the surface of a cell, such as radioisotope labeling, gold labeling, etc., along with transmission electron microscopy.

[0086] In some embodiments, the cells are derived from neural progenitor cells (NPCs). NPCs are progenitor cells of the central nervous system and give rise to many of the neural or glial cells present in the central nervous system. Cells derived from NPCs include, but are not limited to, neurons, cells isolated from primary neuronal tissue, microglia, glial cells, astrocytes, oligodendrocytes, etc., or are derived from neurons. As used herein, "neuron-derived" refers to any cell that is first isolated from neuronal tissue and transformed from a primary neuronal cell to express one or more immortalization factors, such as hTERT, or immortalized cell lines found in neuronal tissue. In some embodiments, the cells are E18 hippocampal neurons. EXAMPLES

[0087] The following examples are illustrative and should not be construed as limiting the scope of the claimed subject matter.

[0088] Example 1 - Effect of NU-9 on AβO binding to Aβ42-treated neurons In neurons treated with amyloid-β peptide, (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione (NU-9) reduces the number of bound amyloid-β oligomers. In Alzheimer's disease model mice, the compound alleviated memory loss. NU-9 inhibits the accumulation of toxic amyloid-β oligomers as well as other aggregated species.

[0089] NU-9 reduces the number of amyloid-β oligomers bound to neurites. Primary rat hippocampal neurons in cell culture were pretreated with NU-9 for 30 min and then treated with amyloid-β1-42 (Aβ) monomers. After Aβ monomers were applied for 24 h (Figures 1 and 2) or 30 min (Figure 3), pretreatment with this compound dramatically reduced the number of amyloid-β oligomers (AβOs) bound to neurons after 24 h. To characterize the dose-response effect of NU-9 on AβO formation, cells were pretreated with increasing doses of NU-9 (3, 15, and 30 μM) and then treated with Aβ for 30 min. Visual analysis of images showed a more significant reduction in amyloid-β with higher doses of NU-9 (Figure 4).

[0090] In the absence of cells, NU-9 has no effect on AβO formation. When NU-9 was mixed with Aβ monomers for 24 hours in the absence of cells, no change in AβO formation was observed (Figure 5). This indicates that NU-9 acts through a cell-based mechanism. Next, a follow-up experiment was performed using conditions that are closer to those of cell culture. Coverslips were coated with poly-D-lysine, as usual for cell culture, and then placed on culture dishes with medium. No cells were added. Then, 3 μM NU-9 and 500 nM AβO were added for 30 min. In this assay, the previous results were reproduced. Without cells, NU-9 had no effect on AβO formation (Figure 6).

[0091] In preliminary experiments with a mouse model of Alzheimer's disease, NU-9 prevented memory loss. Twelve 5xFAD mice were bred on a delayed-onset background. The animals were gavaged daily with either vehicle control or NU-9 (20 mg / kg) for one month. In the control group, all animals failed the memory task and showed no preference for the novel object. In the treatment group, three out of four mice were protected from memory loss and showed a preference for the novel object (Figure 7).

[0092] Reference materials:

[0093] [ka]

[0094] Example 2 - Study of the mechanism by which NU-9 affects AβO formation and binding to neurons Addition of NU-9 (3 μM, 30 min) to neurons prior to treatment with Aβ monomer (500 nM, 30 min or 24 h) significantly reduced AβOs at synapses compared to treatment with Aβ monomer alone. This result was replicated in three separate experiments. The reduction was 81% (p=0.0005). We further characterized the effect of NU-9 to narrow down potential candidate mechanisms for this effect.

[0095] First, we extended previous experiments showing that the effect of NU-9 requires cells. We observed that pretreatment of cells with NU-9 reduced AβOs bound to dendrites, even when NU-9 was washed out before adding Aβ monomers (Figure 8). In addition, we collected conditioned medium produced by untreated cells and found that adding NU-9 (3 μM, 30 min) to the medium did not reduce the number of AβOs formed from Aβ monomers (500 nM, 30 min) (Figure 9, p=0.4). We also collected conditioned medium produced by cells treated with 3 μM NU-9 for 30 min and found that this medium did not reduce the number of AβOs formed from Aβ monomers in the absence of cells (p=0.06). In dot blot analysis, the addition of NU-9 to Aβ monomers did not directly reduce AβO formation in fresh medium (p 0.85, 3 μM, 30 μM, 0.18, 300 μM) or conditioned medium (p 0.88 NU-9+Aβ, 0.54 for NU-9-derived medium in cells+Aβ) (Figure 10). Based on this experiment, we conclude that NU-9 does not inhibit extracellular AβO formation. Interestingly, NU-9 does not directly interact with Aβ or induce the secretion of Aβ-degrading proteases or induce the secretion of chaperones to stabilize Aβ monomers. Instead, NU-9 acts at the cell membrane or intracellularly.

[0096] A previous publication by Pitt et al. showed that astrocytes confer protection to neurons against AβOs by releasing insulin and insulin-like growth factors, which trigger the release of AβOs from neuronal synapses. 1 However, we previously found that NU-9 reduced AβO binding to neurons treated with Aβ monomers but not precursor AβOs (Figure S11), suggesting that NU-9 does not inhibit AβO binding by inducing AβO release. Furthermore, this suggests that NU-9 does not act by directly interfering with AβO binding or by inducing internalization of bound receptors.

[0097] We next expanded on our results showing that NU-9 does not activate proteasomal degradation of AβOs. We previously found that proteasome inhibition did not increase the number of AβOs produced by Aβ monomer-treated neurons, supporting the hypothesis that proteasome activation may not be the mechanism by which NU-9 acts. To follow up on this, we treated neurons with NU-9 (3 μM, 30 min) followed by amyloid-β monomer (500 nM, 30 min) and observed total amyloid-β species by dot blot. However, the total Aβ antibody (6E10) showed nonspecific interactions with the cell culture medium. Therefore, instead of using dot blot, we used immunofluorescence to label neurons for total amyloid-β species. Using this protocol, we were able to avoid interactions between the antibody and the cell culture medium, allowing for specific labeling. Using immunofluorescence, we found that NU-9 did not reduce the total amyloid-β species observed in neuronal cultures (Figure 12, p=0.9 for particles, p=0.4 for IntDen).

[0098] Effect of lysosomal inhibition on the efficacy of NU-9. Cells were first treated with lysosomal inhibitor (100 nM bafilomycin A, 30 min), then with NU-9 (3 μM, 30 min), and finally with Aβ monomer (500 nM, 30 min). AβO puncta bound to dendrites were then quantified. NU-9 treatment in the absence of lysosomal inhibitor reduced the formation of neuron-associated AβO (p=2.6E-5), whereas NU-9 treatment in the presence of lysosomal inhibitor failed to do so (p=7.0E-6) (Figure 13). Furthermore, treatment with Aβ and lysosomal inhibitors in the absence of NU-9 did not result in the formation of significantly more neuron-associated AβO compared to treatment with Aβ monomer alone, suggesting that lysosomal inhibition does not increase AβO but only acts to inhibit the effect of compound NU-9.

[0099] These results suggest that NU-9 acts on a mechanism by which lysosomes promote the formation of unbound AβOs instead of neuron-bound AβOs, possibly through enhanced lysosomal acidification. Paredes-Rosan et al. showed that at lower pH (≦5), lower molecular weight AβOs are more stable than higher molecular weight AβOs. 3 In general, high molecular weight oligomers are found to bind strongly to neurons, whereas low molecular weight oligomers are unable to do so. 4 Thus, lysosomal acidification may be a rational mechanism for the effect of NU-9 on the formation of neuron-associated AβOs (Figure S14).

[0100] In the future, Lysotracker or fluorescent lysosomal substrates will be used to measure potential changes in lysosomal activity induced by NU-9. To address the effect of NU-9 on AβO-induced loss of synaptic spines, the previously established synaptic spine assay will be optimized by testing the responsiveness of cells to AβO-induced toxicity by co-staining for markers of cellular vulnerability or potentially by using other synaptic markers such as drebrin antibodies. The spine assay will then be used to determine the effect of NU-9 on AβO-induced toxicity.

[0101] References:

[0102] [ka]

[0103] Example 3 - Characterization of the lysosome-dependent mechanism by which NU-9 suppresses neuron-associated AβO formation In this further example, the authors' focus was to characterize the lysosomal mechanism by which addition of NU-9 (3 μM, 30 min) to neurons prior to Aβ monomer (500 nM, 30 min or 24 h) causes a 55-80% reduction in AβOs at synapses compared to treatment with Aβ monomer alone (p = 0.001-0.0005 across three experiments). Experiments showing, first, that this effect was maintained when NU-9 was washed out prior to Aβ monomer addition, second, that in cell-free medium, NU-9 did not reduce the number of AβOs formed from Aβ monomers (p = 0.4), and third, that conditioned medium produced by cells treated with NU-9 did not reduce the number of AβOs formed from Aβ monomers in the absence of cells (p = 0.06) support the conclusion that the mechanism of this effect is located intracellularly or at the cell membrane.

[0104] Furthermore, NU-9 did not reduce the total amyloid-β species observed in neuronal cultures (p = 0.4), and treatment of neurons with NU-9 prior to application of amyloid-β monomers to neurons did not reduce the number of unbound extracellular AβOs, but actually increased the number of unbound amyloid-βOs two-fold (p = 0.003), suggesting that NU-9 acts by altering the formation of neuron-bound amyloid-βO species or by removing amyloid-βO-bound targets from the cell surface without promoting the degradation of amyloid-β monomers or oligomers. Finally, the effect of NU-9 required lysosomal acidification and was abolished in the presence of a lysosomal acidification inhibitor (bafilomycin A, p = 7.0E-6). All these results support the hypothesis that NU-9 suppresses the formation of neuron-bound AβO species via a lysosome-dependent mechanism. This mechanism may be directly attributable to the low pH at which more toxic and high molecular weight AβOs are unstable1-3. Alternatively, it may be due to the activation of lysosomal cathepsins by NU-9, which also affect Aβ aggregation and are most active when the lysosomal pH is low 4–7.

[0105] We first verified that NU-9 does not affect the binding of preformed AβOs to neurons. If NU-9 had any effect on preformed AβOs, we hypothesized that NU-9 could bind NKAα3 or PRP. c AβO binding targets such as the neuronal membrane 8-10 This would support the alternative hypothesis that NU-9 removes AβOs from the nucleoids. In these experiments, visual and preliminary qualitative analysis confirmed that NU-9 (3 μM, 30 min, 30 min) did not affect the binding of preformed AβOs (200 and 500 nM, 30 min, FIG. 15). These experiments further support the conclusion that NU-9 not only prevents the binding of preformed, binding-competent AβOs, but also inhibits the formation of specific species of neuron-associated AβOs.

[0106] We also verified experimental findings indicating that the effect of NU-9 depends on lysosomal acidity. We observed a replication of this result in the current experiments (Figure 16). As before, NU-9 (3 μM, 30 min pretreatment) significantly reduced AβOs bound to neurons treated with Aβ monomers (500 nM, 30 min), and the presence of a lysosomal acidification inhibitor (bafilomicin A, 100 nM, 30 min prior to NU-9) suppressed the effect of NU-9 (p2.55E-5, 0.008). This result highlights the importance of lysosomal acidity for the function of NU-9.

[0107] To test whether the lysosomal-dependent effects of NU-9 depend on the function of a key lysosomal enzyme, cysteine ​​cathepsin, a cysteine ​​cathepsin inhibitor was used. First, mature hippocampal neurons were treated with the cysteine ​​cathepsin inhibitor E64 (10 μM, 24 h), then with NU-9 (3 μM, 30 min), and finally with Aβ monomers (500 nM, 30 min). As expected, NU-9 significantly reduced the formation of neuron-bound AβOs (p 0.005). E64 did not significantly alter the efficacy of NU-9 (p 0.8, Figure 17). Unusually, E64 alone also significantly reduced the number of bound AβOs (p 0.01). The effects of E64 and NU-9 were not additive, suggesting that they act within the same mechanism. However, the dependence of NU-9 on lysosomal acidity suggests that it is also not a cysteine ​​cathepsin inhibitor. Based on this result, NU-9 may be an activator of specific cysteine ​​cathepsins, acting by a different mechanism on the same class of targets as E64. It is also possible that NU-9 simply activates lysosomal acidification or endosomal uptake of Aβ for delivery to lysosomes.

[0108] To determine whether NU-9 enhances endolysosomal acidity, we also began optimizing a lysosomal activity assay using Lysotracker, a fluorescent dye that preferentially takes up into acidic compartments and fluoresces more strongly with increasing acidity, and is commonly used to assay lysosomal activation. 11-14 We found that a clear lysosomal activity-dependent signal was observed in mature neurons using 75 nM, 30 min incubation with Lysotracker (Figure 18). The intensity of the lysosomal activity-dependent signal was also low using 50 nM, 2 h incubation with Lysotracker. Fixing the cells before imaging resulted in severe loss of signal. To complete optimization of Lysotracker, we will determine whether lysosomal activation is observed using the lysosomal activator tolquinib.

[0109] Another area of ​​research this quarter was the effect of NU-9 on the formation of different sizes of AβOs. Evidence from animal models and human samples indicates that some species of AβOs are more toxic than others, and that high molecular weight oligomers (>50 kDa) may contain the most AD-associated species. 15-16 To investigate whether NU-9 reduces the formation of high molecular weight AβOs, several experiments were performed using molecular weight cutoff filters of 50 and 100 kDa. However, conflicting results were obtained from different blots. In one experiment, a decrease in the 50–100 kDa species was observed, accompanied by an increase in the small species (<50 kDa). In another experiment, a decrease was observed in most species, except for the small species (<50 kDa). In these experiments, sufficient biological replicates and thorough mixing are important. These experiments may be revisited in the future, and the question of which type of AβO to target may be resolved by proteomics.

[0110] References:

[0111] [ka]

[0112] [ka]

[0113] [ka]

[0114] Example 4 - NU-9 acts via a lysosome-dependent mechanism In this example, we further explore the mechanism of action of NU-9 to prevent AβO formation and accumulation on neurons.

[0115] NU-9 did not affect AβO binding to hippocampal neurons, supporting the conclusion that NU-9 does not prevent binding of preformed AβOs but rather interferes with distinct cellular mechanisms of AβO formation and accumulation (Figure 19).

[0116] In this experiment, we observed that NU-9 reduced the number of AβOs bound to dendrites, even when NU-9 was washed away before application of Aβ monomers, supporting the cellular mechanism of action of NU-9 (Figure 20).

[0117] NU-9 did not alter the number of unbound extracellular AβOs, suggesting a specific effect on the accumulation of toxic cell-bound species (FIG. 21).

[0118] Proteasome inhibition with 100 nM MG132 did not affect AβO formation or the reduction of AβO accumulation by NU-9, suggesting that NU-9 does not act in a proteasome-dependent manner (Figure 22).

[0119] Inhibition of lysosomal maturation and exocytosis with vacclorin-1 prevented the efficacy of NU-9, supporting the conclusion that NU-9 acts through a lysosome-dependent mechanism (Figure 23).

[0120] After 30 min of NU-9 treatment, the integrated fluorescence density of Lysotracker, indicative of acidic lysosomes, did not change, but was significantly decreased by addition of the control bafilomycin A. Lysosomal number, size, and acidity were not altered by NU-9. These results support the conclusion that NU-9 does not act by altering lysosomal number or acidity (Figure 24).

[0121] Addition of 10 μM CA-074, a cathepsin B inhibitor, to neurons for 24 h prior to addition of NU-9 prevented the effect of NU-9, supporting the conclusion that the effect of NU-9 in reducing AβO accumulation in neurons is cathepsin B dependent (Figure 25).

[0122] No effect of 0.0003–150 μM NU-9 was observed on cathepsin L activity, as measured by the rate of production of 7-AMC from Z-FR-AMC by purified cathepsin L enzyme. In this assay, the standard inhibitor Z-FY-CHO reduced cathepsin L activity over the same concentration range. This result supports the conclusion that NU-9 does not directly inhibit cathepsin L, and NU-9 was also not observed to modulate intracellular cathepsin L activity measured in hippocampal neuronal cultures (Figure 26).

[0123] Inhibition of cathepsin L mimics the effects of NU-9 to prevent AβO accumulation. Neurons were pretreated first with 10 μM of the cathepsin L inhibitor Z-FY-CHO (aka SB-412515, 10 μM) for 1 h, then with 3 μM of NU-9 for 30 min, and finally with ~500 nM Aβ for 30 min. Cathepsin L inhibitor treatment mimicked the effects of NU-9 (p<0.0001) (Figure 27). This trend was reproduced in follow-up experiments using a separate cell culture.

[0124] A minimal effect of NU-9 (0.0003-30 μM) on the activity of purified cathepsin B enzyme (cleavage of Z-RR-AMC to 7-AMC) was observed. A clear inhibition of enzyme activity was observed over the same concentration range of the standard cathepsin B inhibitor CA-074. This result supports the conclusion that NU-9 may not directly activate cathepsin B. In cell-based assays, NU-9 was not observed to alter cathepsin B activity in hippocampal neurons (Figure 28).

[0125] Calpain inhibition may mimic the effects of NU-9. To test the effect of calpain inhibition on the efficacy of NU-9 and AβO accumulation, we used the specific inhibitor MDL-28170. 10 μM MDL-28170 was applied to mature hippocampal neurons for 30 min, followed by the addition of 3 μM NU-9, and finally the introduction of 500 nM Aβ42. Inhibition of calpain was observed to also reduce AβO accumulation, mimicking the effect of NU-9 (p<0.0001) (Figure 29). The reduction of AβO accumulation by both calpain and cathepsin L supports the conclusion that AβO accumulation is dependent on the activity of these intracellular cysteine ​​cathepsins.

[0126] This example also demonstrates the ability to detect candidate compounds that modulate amyloid-β oligomerization by comparison with NU-9.

[0127] In conclusion, we discovered that NU-9 does not reduce AβO toxicity by preventing AβO binding to neurons, but rather NU-9 acts through a cellular lysosome-dependent mechanism to prevent AβO toxicity.

[0128] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein can be implemented in the absence of any element, element, restriction or limitation not specifically disclosed herein. The terms and expressions that have been used are used as terms of description and not as terms of limitation, and there is no intention in the use of such terms and expressions to exclude the equivalents of the illustrated and described features or parts thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, although the invention has been described by specific embodiments and optional features, it should be understood that modifications and / or variations of the concepts disclosed herein may be utilized by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0129] Citations to numerous patent and non-patent literature may be made herein. The cited references are incorporated herein by reference in their entirety. If there is a discrepancy between the definition of a term in a cited reference and the definition of a term in the specification, the term shall be construed in accordance with the definition in the specification.

Claims

Claim 1 A method for treating Alzheimer's disease in a subject in need thereof, comprising administering to the subject an effective amount of (S)-5-(1-(3,5-bis(trifluoromethyl)phenoxy)ethyl)cyclohexane-1,3-dione or a suitable pharmaceutical salt thereof to treat Alzheimer's disease in the subject.

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