CN100536837C - Use of scyllo-inositol for preparing diagnostic reagent - Google Patents

Abstract

Disclosed herein are methods of preventing, treating or diagnosing a disorder of protein folding or accumulation, or amyloid formation, deposition, accumulation or persistence in a subject, comprising administering to the subject a pharmaceutically effective amount of an inositol stereoisomer, enantiomer or derivative thereof.

Description

Use of scyllo-inositol in the preparation of diagnostic reagents

related application

This application claims priority to US Provisional Patent Applications Serial Nos. 60/451,363, 60/520,958, and 60/523,534, filed February 27, 2003, November 17, 2003, and November 19, 2003, respectively.

field of invention

The present invention relates to methods of treating Alzheimer's disease and other amyloidoses; more particularly, the present invention relates to inhibition and reduction of amyloidogen in therapeutic interventions for Alzheimer's disease and other amyloidoses The method of fiber formation.

Description of related technologies

The neuropathological hallmarks of Alzheimer's disease are amyloid deposits, neurofibrillary tangles, and selective neuronal loss. The major component of amyloid deposits is amyloid-β (Aβ), which is a 39-43 residue peptide. The soluble form of Aβ produced by cleavage of the amyloid precursor protein is a normal product of metabolism. The importance of residues 1-42 (Aβ42) in Alzheimer's disease is highlighted by the discovery that mutations at codon 717 of the amyloid precursor protein gene, presenilin 1 and presenilin 2 genes lead to a greater ratio of Aβ42 to Aβ1-40 Output increases. These results relating to the presence of Aβ42 in mature plaques and diffuse amyloid lead to the hypothesis that this more amyloid-producing substance may be a critical factor in plaque formation. This hypothesis is supported by the fact that Aβ42 deposition exceeds Aβ40 deposition in PS1 mutated Down syndrome and hereditary cerebral hemorrhage with amyloidosis.

Numerous in vitro studies have demonstrated that Aβ can be neurotoxic or can increase the susceptibility of neurons to excitotoxic, metabolic or oxidative damage. Initially, only the fibrillar form of A was thought to be toxic to neurons, but more comprehensive characterization of the Aβ structure confirmed that Aβ dimers and small aggregates are also neurotoxic. These data suggest that preventing Aβ oligomerization would be an appropriate strategy to prevent neurodegeneration associated with AD. Several studies have demonstrated that Aβ-induced neurotoxicity can be eliminated in vitro using compounds that increase neuronal resistance by targeting cellular pathways involved in apoptosis, blocking downstream pathways following Aβ induction that disrupts pathways, or Blocking Aβ oligomerization and eventual fibril formation increases neuronal resistance. Although the site of A[beta] responsible for inducing neurotoxicity remains to be elucidated, its toxic effects have been blocked by various agents.

Docking of Aβ-fibrils to neuronal and glial cell membranes may be an early interventional step in the AD process. The formation of amyloid plaques, neurotoxicity, and inflammation may be the direct or indirect result of molecular interactions between A and sugar-containing groups. Earlier studies have demonstrated that the interaction of Aβ with aminopolysaccharides leading to Aβ aggregation may increase its insolubility and plaque persistence. Aminopolysaccharides have also been linked to neuronal toxicity and microglial activation. Additionally, interactions with glycolipids such as gangliosides lead to stabilization and prevention of Ab fibril formation and Aβ production sites. On the other hand, the phosphatidylinositol family leads to accelerated fibril formation. The head group of phosphatidylinositol is inositol, a naturally occurring simple sugar involved in lipid biosynthesis, signal transduction, and control of osmotic pressure.

It is also worth noting that amyloid deposition has been demonstrated in a variety of other human diseases and often involves systemic organs (i.e., organs or tissues located in the periphery of the central nervous system) with amyloid leading to organ dysfunction or failure build up. For Alzheimer's disease and "systemic" amyloid diseases, there is currently no cure or effective treatment, and patients typically die within 3-10 years of onset.

US Patent No. 4,847,082 discloses the use of phytic acid, phytic acid salts, isomers or hydrolysates of phytic acid for the treatment of Alzheimer's disease. Also disclosed are isomers of phytic acid or phytates comprising myo-inositol hexaphosphate, scyllo-inositol hexaphosphate, D-chiro-inositol hexaphosphate, L-chiro-inositol hexaphosphate, L-chiro-inositol hexaphosphate, Alcohol ester, neo-inositol hexaphosphate and muco-inositol hexaphosphate configuration. Phytic acid is inositol-hexaphosphate (IP6).

US Patent No. 5,112,814 discloses the use of phytic acid and its isomers for the treatment of Parkinson's disease. Same as US Patent No. 4,847,082, the patent discloses phytic acid isomers having six phosphate groups on the six-carbon inositol sugar.

Notably, in subsequent publications, inositol-monophosphate, inositol-1,4-bisphosphate, and inositol-1,4,5-triphosphate were studied to inhibit amyloid-β peptide ability to generate microfibrils and found them ineffective (J. Mol. Biol. 278:183-194, 1998).

Barak et al. disclose the use of inositol for the treatment of Alzheimer's disease (AD). (Prog Neuro-psychopamacol & Biol Psychiat. 20:729-735, 2000). However, this reference does not disclose the use of inositol isomers. Between myo-inositol-treated AD patients and placebo (glucose)-treated AD patients, myo-inositol-treated patients showed no significant difference in overall cognitive function scores (CAMCOG index), but two specific subgroups of the CAMCOG index Indicators showed significant improvement (orientation and language).

Levine J. reviewed the aforementioned Barak et al. article and specifically pointed out that inositol treatment is not beneficial for AD or ECT-induced cognitive deficits (Eur Neuropsychoparm. 1997; 7, 147-155, 1997).

Referring to the aforementioned article by Barak et al., Colodny et al. further research suggested that myo-inositol is useless for Alzheimer's disease and therefore did not disclose or suggest the use of myo-inositol isomers (Altern Med Rev 3(6):432- 47, 1998).

McLaurin et al. disclosed that myo-inositol can stabilize small micelles of A[beta]42 (J. Mol. Biol. 278, 183-194, 1998). In addition, McLaurin et al. disclosed that epi-inositol and scyllo-inositol, but not chiral-inositol, can induce the structural transformation of Aβ42 from random to β-structure (J Biol Chem. Jun 16;275(24):18495 -502, 2000; and J Struct Biol 130:259-270, 2000). In addition, none of the stereoisomers induced structural transformation of Aβ40. Electron microscopy revealed that inositol stabilizes small accumulations of Aβ42. These references also disclose that inositol-A[beta] interaction produces a complex that is nontoxic to nerve growth factor differentiated PC-12 cells and primary human neuronal cultures.

Much work has been done on Alzheimer's disease, but few compounds or agents are currently known to be used in therapeutic regimens to prevent or reverse the amyloid formation that occurs in Alzheimer's disease or other amyloidoses , deposition, accumulation and/or retention.

Therefore, there is a great need for new compounds or agents for use in therapeutic regimens to prevent or reverse amyloid formation, deposition, accumulation and/or persistence that occur in Alzheimer's disease or other amyloidoses.

Summary of the invention

The present invention provides a method of treating or preventing central or peripheral nervous system or systemic organ disorders related to protein folding or accumulation disorders, or amyloid formation, deposition, accumulation or persistence in a subject, comprising adding the The subject is administered a pharmaceutically effective amount of a compound selected from the following structures:

Wherein, each R ₁ , R _1' , R ₂ , R _2' , R ₃ , R _3' , R ₄ , R _4' , R ₅ , R _5' , R ₆ and R _6' are independently selected from the following groups group:

(a) a hydrogen atom;

(b) NHR ₇ , wherein said R ₇ is selected from hydrogen; C ₂ -C ₁₀ acyl and C ₁ -C ₁₀ alkyl;

(c) NR ₈ R ₉ , wherein said R ₈ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl, and said R ₉ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl;

(d) OR ₁₀ , wherein said R ₁₀ is selected from the group consisting of no group, hydrogen, C ₂ -C ₁₀ acyl, C ₁ -C ₁₀ alkyl and SO ₃ H;

(e) C ₅ -C ₇ sugar groups;

(f) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OH, NH ₂ , SH, OSO ₃ H and OPO ₃ H ₂ ;

(g) SR ₁₁ , wherein R ₁₁ is selected from hydrogen, C ₁ -C ₁₀ alkyl and O ₃ H;

(h) C ₁ -C ₁₀ alkyl optionally substituted with a substituent selected from hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ; and

(i) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ,

Provided that the compound is not myo-inositol.

The present invention also provides a method of preventing abnormal protein folding, abnormal protein accumulation, amyloid formation, deposition, accumulation or retention, or amyloid lipid interaction in a subject, comprising administering to the subject A pharmaceutically effective amount of a compound selected from the following structures:

Wherein, each R ₁ , R _1' , R ₂ , R _2' , R ₃ , R _3' , R ₄ , R _4' , R ₅ , R _5' , R ₆ and R _6' are independently selected from the following groups group:

(a) a hydrogen atom;

(b) NHR ₇ , wherein said R ₇ is selected from hydrogen; C ₂ -C ₁₀ acyl and C ₁ -C ₁₀ alkyl;

(c) NR ₈ R ₉ , wherein said R ₈ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl, and said R ₉ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl;

(d) OR ₁₀ , wherein said R ₁₀ is selected from the group consisting of no group, hydrogen, C ₂ -C ₁₀ acyl, C ₁ -C ₁₀ alkyl and SO ₃ H;

(e) C ₅ -C ₇ sugar groups;

(f) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OH, NH ₂ , SH, OSO ₃ H and OPO ₃ H ₂ ;

(g) SR ₁₁ , wherein R ₁₁ is selected from hydrogen, C ₁ -C ₁₀ alkyl and O ₃ H;

(h) C ₁ -C ₁₀ alkyl optionally substituted with a substituent selected from hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ; and

(i) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ,

Provided that the compound is not myo-inositol.

The present invention also provides a method of dissociating abnormally accumulated protein and/or dissolving or disrupting preformed or predeposited amyloid fibrils or amyloid in a subject comprising administering to said subject Administration of a pharmaceutically effective amount of a compound selected from the following structures:

Wherein, each R ₁ , R _1' , R ₂ , R _2' , R ₃ , R _3' , R ₄ , R _4' , R ₅ , R _5' , R ₆ and R _6' are independently selected from the following groups group:

(a) a hydrogen atom;

(b) NHR ₇ , wherein said R ₇ is selected from hydrogen; C ₂ -C ₁₀ acyl and C ₁ -C ₁₀ alkyl;

(c) NR ₈ R ₉ , wherein said R ₈ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl, and said R ₉ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl;

(d) OR ₁₀ , wherein said R ₁₀ is selected from the group consisting of no group, hydrogen, C ₂ -C ₁₀ acyl, C ₁ -C ₁₀ alkyl and SO ₃ H;

(e) C ₅ -C ₇ sugar groups;

(f) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OH, NH ₂ , SH, OSO ₃ H and OPO ₃ H ₂ ;

(g) SR ₁₁ , wherein R ₁₁ is selected from hydrogen, C ₁ -C ₁₀ alkyl and O ₃ H;

(h) C ₁ -C ₁₀ alkyl optionally substituted with a substituent selected from hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ; and

(i) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ,

Provided that the compound is not myo-inositol.

The present invention also provides a method of diagnosing the presence of abnormally folded or accumulated proteins and/or amyloid fibrils or amyloid in a subject, comprising: (a) administering to said subject a radioactive compound or Compounds labeled with a substance that emits a detectable signal under conditions that permit binding of said compound to abnormally folded or accumulated proteins and/or fibrils or amyloids, if the abnormally folded or accumulated proteins and/or fibrils or amyloid, if present; and (b) detecting radioactivity or a signal from a compound that binds to the abnormally folded or accumulated protein and/or fibrils or amyloid, thereby diagnosing the subject for abnormal folding or accumulation Presence of protein and/or amyloid fibrils or amyloid, wherein said compound has the following structure:

Wherein, each R ₁ , R _1' , R ₂ , R _2' , R ₃ , R _3' , R ₄ , R _4' , R ₅ , R _5' , R ₆ and R _6' are independently selected from the following groups group:

(a) a hydrogen atom;

(b) NHR ₇ , wherein said R ₇ is selected from hydrogen; C ₂ -C ₁₀ acyl and C ₁ -C ₁₀ alkyl;

(c) NR ₈ R ₉ , wherein said R ₈ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl, and said R ₉ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl;

(d) OR ₁₀ , wherein said R ₁₀ is selected from the group consisting of no group, hydrogen, C ₂ -C ₁₀ acyl, C ₁ -C ₁₀ alkyl and SO ₃ H;

(e) C ₅ -C ₇ sugar groups;

(f) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OH, NH ₂ , SH, OSO ₃ H and OPO ₃ H ₂ ;

(g) SR ₁₁ , wherein R ₁₁ is selected from hydrogen, C ₁ -C ₁₀ alkyl and O ₃ H;

(h) C ₁ -C ₁₀ alkyl optionally substituted with a substituent selected from hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ; and

(i) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ,

Provided that the compound is not myo-inositol.

The present invention also provides a method for diagnosing the presence of abnormally folded or accumulated proteins and/or amyloid fibrils or amyloid in a subject, comprising: (a) collecting a sample from the subject; ( b) contacting said sample with a radioactive compound or a compound labeled with a substance emitting a detectable signal under conditions allowing said compound to bind to abnormally folded or accumulated proteins and/or amyloid fibrils or amyloid, If the abnormally folded or accumulated protein and/or amyloid fibrils or amyloid are present; and (c) detecting radioactivity or signal, thereby diagnosing the presence of abnormally folded or accumulated proteins and/or amyloid fibrils or amyloid in said subject, wherein said compound has the following structure:

Wherein, each R ₁ , R _1' , R ₂ , R _2' , R ₃ , R _3' , R ₄ , R _4' , R ₅ , R _5' , R ₆ and R _6' are independently selected from the following groups group:

(a) a hydrogen atom;

(b) NHR ₇ , wherein said R ₇ is selected from hydrogen; C ₂ -C ₁₀ acyl and C ₁ -C ₁₀ alkyl;

(c) NR ₈ R ₉ , wherein said R ₈ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl, and said R ₉ is C ₂ -C ₁₀ acyl or C ₁ -C ₁₀ alkyl;

(d) OR ₁₀ , wherein said R ₁₀ is selected from the group consisting of no group, hydrogen, C ₂ -C ₁₀ acyl, C ₁ -C ₁₀ alkyl and SO ₃ H;

(e) C ₅ -C ₇ sugar groups;

(f) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OH, NH ₂ , SH, OSO ₃ H and OPO ₃ H ₂ ;

(g) SR ₁₁ , wherein R ₁₁ is selected from hydrogen, C ₁ -C ₁₀ alkyl and O ₃ H;

(h) C ₁ -C ₁₀ alkyl optionally substituted with a substituent selected from hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ; and

(i) C ₃ -C ₈ cycloalkyl, which is optionally substituted by a substituent selected from the group consisting of hydrogen, OR ₁₀ , NHR ₇ , NR ₈ R ₉ and SR ₁₁ ,

Provided that the compound is not myo-inositol.

Brief introduction to the drawings

Figure 1A presents the structures of myo-inositol, epi-inositol, and scyllo-inositol, while Figures 1B-1H show spatially referenced memory models for the Morris water maze test performed on TgCRND8 mice. Myo-inositol treatment did not alter cognitive function (1B). At 6 months of age, untreated TgCRND8 (n=10) showed cognitive deficits relative to non-Tg control (1C) and scyllo-inositol (1D) treated mice (n=10 each, untreated Compared with the treatment group p<0.02). Performance of epi-inositol-treated TgCRND8 mice was still impaired compared to non-Tg littermates (1E), but performance of scyllo-inositol TgCRND8 mice was similar to that of non-Tg littermates ( 1F). Behavior of non-Tg littermates was not affected by epi-inositol (1G) or scyllo-inositol (1H) treatment. Vertical bars represent standard errors.

Figures 2A-2I show plaque accumulation and astrogliosis in epi-inositol and scyllo-inositol treated TgCRND8 at 6 months of age. Control animals had high plaque burden and astrogliosis in the hippocampus (2A) and cerebral cortex (2B). Higher magnification demonstrates that astroglial activation is not solely related to plaque burden (2C). Epi-inositol treatment had a moderate effect on amyloid accumulation and reduced astrogliosis (2D, 2E, and 2F). Scyllo-inositol treatment significantly reduced amyloid accumulation and gliosis (2G, 2H and 2I). Higher magnification shows that scyllo-inositol treated mice have smaller mean plaque sizes (2I). Astrocytes were labeled with anti-GFAP antibody and plaque accumulation was identified using anti-Aβ antibody. Scale bars are 450 microns (A, B, D, E, G, H) and 94 microns (C, F, I).

Figures 3A-3D show that Aβ species 1-42, 1-40 and 1-38 are indistinguishable from the extent of APP progression (3B) in control and treated TgCRND8 mice. Vascular amyloid accumulation was quantified in serial sagittal sections from treated and untreated TgCRND8 mice. TgCRND8 mice had significant vascular amyloid accumulation associated with small and medium sized vessels, and scyllo-inositol-treated TgCRND8 mice had a decreased load (3A). Scyllo-inositol treatment significantly reduced total vascular burden compared to untreated and epi-inositol-treated TgCRND8 mice (3C). A significant reduction in mean plaque size (3D) demonstrates that scyllo-inositol reduces plaque deposition.

Figure 4 shows the effect of water on cognitive function in TgCRND8 and non-Tg mice in a 3-day experimental paradigm using the Morris water maze spatially referenced memory model.

Figure 5 shows the effect of scyllo-inositol on cognitive function in TgCRND8 and non-Tg mice in a 3-day experimental paradigm using the spatially referenced memory model of the Morris water maze.

Figure 6 shows the effect of epi-inositol on cognitive function in TgCRND8 and non-Tg mice in a 3-day experimental paradigm using the Morris water maze spatially referenced memory model.

Figure 7 shows the effect of myo-inositol on cognitive function in TgCRND8 and non-Tg mice in a 3-day experimental paradigm using the Morris water maze spatially referenced memory model.

Figure 8 shows the effects of scyllo-inositol, epi-inositol and myo-inositol on TgCRND8 cognitive function (learning period and memory test) in the 3-day test paradigm using the Morris water maze spatially referenced memory model and compared with wild type mice were compared.

Figure 9 shows the percentage of brain area covered by plaques in untreated TgCRND8 mice compared to scyllo-inositol, epi-inositol or myo-inositol treated mice.

Figures 10A and 10B show the survival rate of TgCRND8 mice treated with water compared with the survival rate of TgCRND8 mice treated with epi-inositol or myo-inositol (10A) or the survival rate of TgCRND8 mice treated with scyllo-inositol Compare (10B).

Figures 11A-D show the results of the spatially referenced memory model of the Morris water maze test in 6-month-old TgCRND8 mice treated or untreated with mannitol (A, B). Mannitol-treated TgCRND8 mice were not significantly different from untreated TgCRND8 mice (p=0.89; A). The performance of mannitol-treated TgCRND8 mice was significantly different from that of mannitol-treated non-Tg littermates (p=0.05; B). Protein accumulation was analyzed using quantitative image analysis (C). Mannitol-treated TgCRND8 mice were indistinguishable from untreated TgCRND8 mice when measured by plaque count as total plaque inventory (p=0.87). Vertical bars represent standard errors. Kaplan-Meier cumulative survival of mannitol-treated or untreated TgCRND8 mice is plotted (D). There was no significant difference between the two groups of animals (n=35 in each group) by Tarone-Ware statistical test, p=0.87.

Figures 12A and B show the results of the treatment study for the spatial reference memory test for the 3-day test paradigm. Performance of scyllo-inositol-treated TgCRND8 mice was comparable to scyllo-inositol-treated non-Tg littermates (p=0.38; A). Consistently, scyllo-inositol-treated TgCRND8 mice remained indistinguishable from non-Tg littermates after 2 months of treatment (p=0.67; B).

Figures 13A and B show the Aβ content in the CNS of 5-month-old TgCRND8 mice administered once daily with various doses of scyllo-inositol for 1 month. At all doses, soluble A[beta]42 levels decreased and were significantly different from untreated controls (A). In contrast, insoluble A[beta]42 was not significantly different under all conditions (B). Vertical bars represent standard errors.

Fig. 14 Analysis of brain Aβ40 content after administration of various doses of scyllo-inositol to TgCRND8 mice once a day for 1 month. At all doses tested, no difference was detected between soluble (A) and insoluble (B) Aβ40 levels in untreated and scyllo-inositol-treated TgCRND8 mice.

Figure 15 shows cognitive function in iso-inositol treated 6-month-old TgCRND8 mice compared to non-transgenic littermates.

Figures 16A-D show that scyllo-inositol treated mice had reduced plaque accumulation in TgPS1 x APP mice at 2 months of age. Control animals had high plaque burden in the hippocampus (A) and cerebral cortex (B). Scyllo-inositol treatment significantly reduced amyloid accumulation (C, D). Plaque accumulation was identified using anti-Aβ antibody (brown). The calibration bar is 300 μm.

Figures 17A-C show quantification of plaque accumulation in TgPS1 x APP mice following scyllo-inositol treatment. Significant reduction in percent plaque brain coverage (A), mean plaque size (B) and plaque count (C). Vertical bars represent standard errors.

Detailed description of the invention

The present invention discloses novel, unpredictable and unexpected properties of certain inositol stereoisomers that are relevant to the treatment of amyloid-related diseases such as Alzheimer's disease.

It has surprisingly been found that certain stereoisomers of myo-inositol and related compounds block Aβ-induced progressive cognitive decline and cerebral amyloid plaque pathology and, when administered to humans during the nascent phase of Aβ deposition, Improved survival in a transgenic mouse model of Alzheimer's disease.

As disclosed above, previous data suggest that some (not all) inositol stereoisomers may play a role in amyloid aggregation of neuronal cells cultured in vitro (McLaurin et al., J. Biol. Chem. 275( 24): 18495-18502 (2000)). Those surveys did not provide any means of predicting which of the studied stereoisomers (myo-inositol, epi-inositol, scyllo-inositol, and chiro-inositol) would have this effect, nor any other will have this effect. Also, those studies could not predict whether any one isomer of inositol would have an effect on amyloid deposition, cognitive deficits, or longevity in vivo. The present invention describes the unpredictable result that only certain stereoisomers of inositol, in particular scyllo-inositol and iso-inositol, reduce amyloid plaque burden in animal models of amyloid-associated disease , improved cognition and increased lifespan, while the other isomers studied did not have such effects.

Previous studies have only suggested that certain inositol stereoisomers (such as epi-inositol and scyllo-inositol) can inhibit nascent amyloid aggregation in vitro. The present invention describes the unpredictable result that scyllo-inositol inhibits established brain amyloid deposits, and also in living brains. This is not suggested by previously published in vitro data, which suggest that it acts only on certain neuronal cell types in culture, not on the complex organization of the living brain, and only suggests that myo-inositol inhibits nascent aggregation and thus correlates with established disease. irrelevant.

Previous in vitro data also suggested that administration of epi-inositol and scyllo-inositol also affects amyloid Aβ40 content and Aβ42 content. In vivo dosing studies of the present invention revealed the following unexpected results: administration of iso-inositol or scyllo-inositol in particular reduces Aβ42 levels, while having no effect on insoluble Aβ42 and soluble or insoluble Aβ40 levels.

The present investigation revealed changes in glial activity and inflammation that were novel and surprising and could not have been predicted from previously published in vivo data.

The present investigations demonstrating that scyllo-inositol improves lifespan in transgenic model animals is also novel and surprising since no Alzheimer's disease drug has previously been shown to increase survival and extend lifespan in vivo.

Preferably, the compound of the present invention is 1,2,3,4,5,6-cyclohexanehexanol, more preferably selected from cis-, epi-, iso-, sticky-, neo-, shark-, D-hand Chiro- and L-chiro-inositol.

Also preferred is the compound 1,2,3,4,5-cyclohexanepentaol (quercitol), more preferably selected from the group consisting of epi-, vibo-, shark-, iso-, taro-, gala-, cis-, sticky- , neo-, pro-quercitol and its enantiomers.

Also preferred are those selected from the group consisting of cyclohexanetriol, cyclohexanetriol, stereoisomers of cyclohexanetriol, stereoisomers of cyclohexanetriol, enantiomers of cyclohexanetriol and cyclohexanetriol Enantiomers of compounds.

These compounds may also be pentahydroxycyclohexanone or its stereoisomers or enantiomers.

Also preferably, these compounds are inositol monoketones selected from the group consisting of scyllo-inositol monoketone, L-chiro-inositol monoketone-1 and L-epi-inositol monoketone.

Also preferably, these compounds are trihydroxycyclohexanone, or stereoisomers or enantiomers thereof. More preferred is (-)-1-deoxy-syllo-inositol monoketone.

Also preferably, these compounds are pentahydroxycyclohexanone (inositol monoketone), or stereoisomers or enantiomers thereof, more preferably selected from the group consisting of scyllo-inositol monoketone, L-chiral-inositol monoketone -1 and L-epi-inositol monoketone.

Optionally, these compounds are trihydroxycyclohexanone or a stereoisomer or enantiomer thereof, such as (-)-1-deoxy-squalyl inositol monoone.

Also preferably, these compounds are O-monomethyl-cyclohexanol or its stereoisomers or enantiomers, more preferably selected from the group consisting of D-pinitol, L-quinayl alcohol and D-boryl alcohol.

In addition, these compounds may be selected from the group consisting of monoaminocyclohexanepentitol (inositolamine), diaminocyclohexitol (inositol diamine), diaminocyclohexanetriol, stereoisomers thereof, and enantiomers thereof Constructs, and pharmaceutically acceptable salts thereof, such as L-neo-inositolamine, D, L-epi-inositolamine-2, streptomycin and deoxystreptamine.

Also preferably, these compounds are monomercapto-cyclohexylpentaol, or stereoisomers or enantiomers thereof, more preferably 1L-1-deoxy-1-mercapto-8-O-methyl-chiral-muscle alcohol.

The most preferred compounds of the present invention are iso-inositol and scyllo-inositol, of which scyllo-inositol is most preferred. As indicated above, the stereoisomers of inositol of the present invention do not include myo-inositol and also do not include epi-inositol.

These compounds were effective in reversing brain Aβ accumulation and amyloidosis even when administered months after amyloidopathy had already developed.

Accordingly, these compounds find use in the treatment or prevention of disorders of the central or peripheral nervous system or systemic organs associated with disordered protein folding or accumulation, or amyloid formation, deposition, accumulation or persistence in a subject. The compounds are also found to be useful in preventing abnormal protein folding, abnormal protein accumulation, amyloid formation, deposition, accumulation or retention, or amyloid lipid interaction in a subject, as well as dissociation and dissociation of abnormally accumulated proteins in a subject. and/or solubilize or disrupt pre-formed or pre-deposited amyloid fibrils or amyloid.

Preferably, said disorders of the central or peripheral nervous system or systemic organs lead to precipitation of proteins, protein fragments and peptides in the form of β-sheets and/or fibrils and/or aggregates. More preferably, the disorder of the central or peripheral nervous system or systemic organs is selected from the group consisting of: Alzheimer's disease, presenile and senescent; amyloid angiopathy; mild cognitive decline; associated with Alzheimer's disease dementia; tau disease; alpha-synuclein disease; Parkinson's disease; amyotrophic lateral sclerosis; motor neuron disease; spastic paraplegia (paraplagia); Huntington's disease; spinocerebellar ataxia; Friedreich's ataxia; associated with intracellular and/or intraneuronal proteins with polyglutamine, polyalanine, or other duplications caused by pathological expansion of three or four nucleotide units within the corresponding gene Neurodegenerative diseases associated with aggregation of cerebrovascular diseases; Down syndrome; head trauma with posttraumatic amyloid beta peptide accumulation; prion-related diseases; familial British dementia; familial Danish dementia; Alzheimer's disease with spastic ataxia; British type cerebral amyloid angiopathy; Alzheimer's disease with spastic ataxia; Danish type cerebral amyloid angiopathy; familial encephalopathy with neurofilin inclusions ( FENIB); amyloid polyneuropathy; inclusion body myositis caused by amyloid beta peptide; familial Finnish amyloidosis; systemic amyloidosis associated with multiple myeloma; familial Mediterranean fever; chronic infection and inflammation and type 2 diabetes associated with islet amyloid polypeptide (IAPP).

Also preferably, the dementia associated with Alzheimer's disease is vascular or Alzheimer's dementia, and the tau disease is selected from argyrophilic grain dementia, cortical matrix degeneration, dementia pugilistica, with calcification Diffuse neurofibrillary tangles, frontotemporal dementia with parkinsonism, prion-related disorders, Hallerwoden-Spatz disease, myotonic dystrophy, Niemann-Pick type C non-Guam motor neuron disease with neurofibrillary tangles, Pick disease, postencephalitic Parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear Paralysis, subacute sclerosing panencephalitis, and tangle only dementia.

Also preferably, the α-synucleinopathies are selected from the group consisting of dementia with centripetally multilamellar round bodies, multiple system atrophy with glial cytoplasmic inclusions, Chard-Dell syndrome, striatonigral degeneration sex, olivopontocerebellar atrophy, neurodegeneration with type I brain iron accumulation, anosmia, and amyotrophic lateral sclerosis.

Also preferably, motor neuron disease is associated with filamentous or aggregated neurofilaments and/or superoxide dismutase proteins, spastic paraplegia is associated with chaperone and/or tripartite A protein dysfunction, and spinocerebellar Ataxia is DRPLA or Ma-John disease.

-Scheinker病、和变种克罗伊茨费尔特-雅各布病，和淀粉样多神经病是老年淀粉样多神经病或全身性淀粉样变性。Also preferably, the disease associated with prions is selected from the group consisting of Creutzfeldt-Jakob disease, Gerstmann-

-Scheinker disease, and variants Creutzfeldt-Jakob disease, and amyloid polyneuropathy are senile amyloid polyneuropathy or systemic amyloidosis.

More preferably, the disorders of the central or peripheral nervous system or systemic organs include familial or non-familial Parkinson's disease. Most preferably, said disorder of the central or peripheral nervous system or systemic organ is Alzheimer's disease.

Preferably, the compound of the present invention is administered at about 1 mg to about 1 g/kg body weight of a subject, preferably 1 mg to about 200 mg/kg body weight of a subject, more preferably about 10 mg to about 100 mg/kg body weight of a subject, most preferably about 30 mg to A dose of about 70 mg/kg of subject body weight is administered to the subject. Administration can be accomplished by various methods such as: oral (oral pills, oral liquid or suspension), intravenous, intramuscular, intraperitoneal, intradermal, transdermal, subcutaneous, intranasal, sublingual, rectal suppositories or inhalation, with oral administration being most preferred. The compounds of the invention may be administered at various intervals, such as once daily, twice daily, weekly, monthly or continuously.

碱激动剂(例如AF102B(西维美林，EVOXAC)、AF150(S)和AF267B)、抗精神病药(例如氟派啶醇、氯氮平、奥氮平)；抗抑郁药，包括三环抗抑郁剂和血清素重摄取抑制剂(例如舍曲林和西酞普兰Hbr)、基于正调节neprilysin(一种降解Aβ的酶)的基因治疗和/或药物；基于正调节胰岛素降解酶(一种降解Aβ的酶)的基因治疗和/或药物、疫苗、Aβ的免疫治疗和抗体(例如ELAN AN-1792)、他汀类药物和其它降胆固醇药物(例如洛伐他汀和辛伐他汀)、干细胞和其它基于细胞的治疗剂、磷酸化TAU蛋白的激酶(CDK5、GSK3α、GSK3β)抑制剂(例如氯化锂)、或调节Aβ生成的激酶(GSK3α、GSK3β、Rho/ROCK激酶)的抑制剂(例如氯化锂和布洛芬)。Preferably, the compounds of the invention are administered in combination with other therapeutic agents such as β-secretase inhibitors, γ-secretase inhibitors (APP-specific or non-specific), ε-secretase inhibitors (APP- specific or non-specific), other inhibitors of β-sheet aggregation/fibril formation/ADDL production (e.g. Alzheimer’s), NMDA antagonists (e.g. memantine), NSAIDs (e.g. ibuprofen, vitamins), antioxidants (e.g. vitamin E), hormones (e.g. estrogen), nutritional and food supplements (e.g. ginkgo biloba); acetylcholinesterase inhibitors (e.g. donezepil), toxic

Base agonists (eg, AF102B (cevimeline, EVOXAC), AF150(S), and AF267B), antipsychotics (eg, haloperidol, clozapine, olanzapine); antidepressants, including tricyclics Depressants and serotonin reuptake inhibitors (such as sertraline and citalopram Hbr), gene therapy and/or drugs based on upregulation of neprilysin (an enzyme that degrades Aβ); gene therapy and/or drugs for enzymes that degrade Aβ), vaccines, immunotherapy and antibodies for Aβ (such as ELAN AN-1792), statins and other cholesterol-lowering drugs (such as lovastatin and simvastatin), stem cells and Other cell-based therapeutics, inhibitors of kinases (CDK5, GSK3α, GSK3β) that phosphorylate TAU proteins (eg, lithium chloride), or inhibitors of kinases that regulate Aβ production (GSK3α, GSK3β, Rho/ROCK kinases) (eg, lithium chloride and ibuprofen).

We believe that these other therapeutic agents act through different mechanisms and are additive/synergistic to the present invention. In addition, many other therapeutic agents may have mechanism-based effects and/or other side effects that limit dosage or tolerability, at which point they can be administered alone.

Because the compounds of the present invention have the ability to bind amyloid in vivo, discussed in more detail below, the compounds of the present invention are also useful for diagnosing abnormally folded or accumulated proteins and/or amyloidogens in a subject using the following methods the presence of fibrils or amyloid, the method comprising: administering to said subject a radioactive compound or labeled with a radioactive compound in sufficient amount to allow said compound to bind to abnormally folded or accumulated proteins and/or fibrils or amyloid; compounds of substances that emit a detectable signal under conditions in which abnormally folded or accumulated proteins and/or fibrils or amyloids are present; The radioactivity or signal of the bound compound, thereby diagnosing the presence of abnormally folded or accumulated protein and/or amyloid fibrils or amyloid.

Additionally, a sample suspected of containing abnormally folded or accumulated protein and/or amyloid fibrils or amyloid is collected from the subject and is treated with a radioactive compound or labeled with a radioactive compound in a manner that allows the compound to associate with the abnormally folded or accumulated contact with a compound that emits a detectable signal under conditions of protein and/or amyloid fibrils or amyloid binding, if abnormally folded or accumulated proteins and/or amyloid fibrils or amyloid are present; and subsequently detect radioactivity or signals from compounds that bind to abnormally folded or accumulated proteins and/or fibrils or amyloid, thereby diagnosing said subject as having abnormally folded or accumulated proteins and/or amyloid fibrils or the presence of amyloid.

Preferably, said detectable signal is a fluorescent signal or an ELISA signal and said sample is whole blood (including all cellular components) or plasma.

As shown below, in a transgenic mouse model of Alzheimer's disease, administration of the compounds of the present invention at the "late presymptomatic" stage, before the development of overt cognitive deficits and amyloid neuropathology in mice, eliminated Aβ accumulation in the brain, cerebral amyloid plaque deposition, and cognitive decline. Furthermore, these compounds were effective in reversing amyloid deposition and neuropathology even when administered after cognitive deficits and amyloid plaque neuropathology. Importantly, rational design will be guided by the mechanism of action of these compounds based on their ability to modulate the assembly of A[beta] monomers into neurotoxic oligomers and/or fibrils.

Other advantages of the compounds of the present invention include their transport to the CNS by two known transporters and passive diffusion, thereby providing good CNS bioavailability. Second, these compounds are metabolized to glucose. Third, as a class of substances, these compounds generally have a low toxicity profile, and some of them have previously been administered to humans for different purposes.

Embodiment 1-Construction of Alzheimer's mouse model and the method of administering the compound of the present invention

As described by Janus et al. (Nature 408:979-982 (2000)), the TgCRND8 mouse is a strong mutational model of Alzheimer's disease. They express the human amyloid precursor protein (APP695) transgene under the control of a Syrian hamster on a C3H/B6 outbred background with a prion promoter. The human APP695 transgene has two mutation points that cause AD in humans (K670N/M671L and V717F). From about 3 months of age, TgCRND8 mice exhibit progressive spatial learning deficits, accompanied by elevated brain Aβ levels and increased numbers of extracellular amyloid plaques in the brain, both of which are consistent with those seen in human brains with AD Similar (C. Janus et al., Nature 408:979-982 (2000)).

Groups of age- and sex-matched TgCRND8 mice and non-transgenic littermate groups (n=35 each) were either left untreated or dosed at 30 mg/day/mouse from approximately 6 weeks of age as follows Administration of compounds of the invention. Cognitive function, brain Aβ content, brain pathology and survival rate of these mice were subsequently measured at 4 and 6 months of age.

Prevention Research Methods

Mice - Myo-inositol, epi-inositol and scyllo-inositol were fed to the TgCRND8 mice of the test group at an amount of 30 mg/mouse/day. Both groups entered the study at 6 weeks of age and the results were analyzed at 4 and 6 months of age. Body weight, coat characteristics, and cage behavior were monitored. All experiments were performed in accordance with the Canadian Council's Animal Care Guidelines.

Behavioral testing - Following non-spatial pre-training, mice were trained for orientation discrimination for 5 days with 4 tests per day. Behavioral data were analyzed using a mixed model of factorial analysis of variance (ANOVA) with drug or genotype and training period as repeated measures factors.

Brain Amyloid Burden - Brains were removed and one hemisphere was fixed in 4% paraformaldehyde and embedded in paraffin along the mid saggital plane. To generate a systematically uniform set of random sections, 5 μm serial sections were collected transecting the entire hemisphere. Groups of slices separated by 50 mm were used for analysis (10-14 slices/group). Plaques were identified after antigen retrieval with formic acid and incubated with primary anti-Aβ antibody (Dako M-0872) followed by secondary antigen (Dako StreptABCcomplex/horseradish kit). The final product was visualized by DAB counterstaining with hematoxylin. Amyloid plaque burden was assessed using Leco IA-3001 image analysis software connected to a Leica microscope and Hitachi KIP-MU CCD camera. Vascular burden was analyzed similarly, and a stripper was used to measure the diameter of the affected vessel.

Plasma and Brain Aβ Content - Brain hemisphere samples were homogenized in sucrose buffer, then either 0.4% diethylamine/100mM NaCl was added to solubilize Aβ content, or cold formic acid was added to separate total Aβ. After neutralization, samples were diluted and analyzed for Aβ40 and Aβ42 using commercially available kits (BIOSOURCE International). Each hemisphere was analyzed three times, and the mean ± standard deviation was recorded. Protein spot analysis using urea gel for analysis of Aβ species was performed on all sections. Aβ was detected using 6E10 (BIOSOURCE International) and enhanced chemiluminescence (Amersham).

Analysis of APP in the brain - Mouse brain hemisphere samples were homogenized in 20mM Tris pH7.4, 0.25M sucrose, 1mM EDTA and 1mM EGTA, and protease inhibitor cocktail, mixed with 0.4% DEA (diethylamine)/100mM NaCl mixed and spun at 109,000Xg. The APPs content in the supernatant was analyzed by Western blot using mAb 22C11, and the pellet was analyzed using mAb C1/6.1 to determine APP total protein.

Quantification of Gliosis - Five evenly spaced sagittal sections were randomly selected from paraformaldehyde-fixed and frozen hemispheres of treated and control mice. Sections were immunolabeled with anti-mouse GFAP IgG2a (Dako; 1:50 dilution) for astrocytes and with anti-mouse CD68 IgG2b (Dako; 1:50 dilution) for microglia. Digital images were collected using a Coolsnap digital camera (Photometrics, Tuscon, Arizona) mounted on a Zeiss Axioscope 2 Plus microscope. Images were analyzed using Openlab 3.08 image software (Improvision, Lexington MA).

Survival Survey - Survival probability is estimated by the Kaplan-Meier technique and is calculated and analyzed whenever a death occurs, making it suitable for small sample sizes. 35 mice per treatment group were used for viability analysis. Comparisons between treatment groups were documented using the Tarone-Ware test.

Example 2 - Prevention of Cognitive Deficiency

Cognitive function in TgCRND8 mice was assessed using a 5-day experimental paradigm using the spatially referenced memory model of the Morris water maze. A mixed analysis of variance (ANOVA) model was used to analyze the differences between the treated and Data obtained from untreated TgCRND8 mice and from treated and untreated non-Tg littermates (n=10 for all combinations). TgCRND8 mice treated with epi-inositol or scyllo-inositol performed significantly better than untreated TgCRND8 mice (p<0.02; Figure 1C and D). Epi-inositol-treated TgCRND8 mice exhibited a slightly slower learning curve during the first three days of training compared to treated and untreated non-Tg littermates. However, after 4 days of training, there was no statistical difference between epi-inositol-treated TgCRND8 mice and their non-Tg littermates (Fig. 2E). In contrast, there was no difference between scyllo-inositol treated TgCRND8 mice and non-Tg littermates on all days. Thus, both stereoisomers inhibit the development of cognitive deficits, and indeed, scyllo-inositol prevents cognitive deficits to such an extent that scyllo-inositol-treated TgCRND8 mice are indistinguishable from normal mice. degree. This improved performance was not due to nonspecific effects on behavioral, motor, or sensory systems, as epi-inositol and scyllo-inositol treatments had no effect on performance in non-Tg mice (Figures 2G and 2H). This improved performance was also not due to nutritional or caloric effects, as there were no differences in body weight, activity, and coat condition between the treated and untreated groups. Furthermore, treatment with mannitol, a sugar of similar molecular weight, had no effect on behavior. The effect of gender was not significant (p=0.85) between any treatment groups.

Example 3 - Reduction of Brain Aβ Burden and Amyloid Neuropathology

Untreated TgCRND8 mice expressed large amounts of Aβ40 and Aβ42 when the mice were 4 months old (Table 1). At 4 months of age, as described in Example 1, the levels of Aβ40 (soluble and insoluble pools decreased by 43±2%; p≤0.05) and Aβ42 levels (soluble pools decreased 69%, p=0.005; 28% reduction in insoluble pool, p=0.02) both decreased. However, these improvements were not durable, with brain A[beta] levels rising at 6 months of age to levels similar to those observed in untreated TgCRND8 mice (Table 1).

In contrast, at 4 months of age, the scyllo-inositol treated group had a 62% decrease in total brain A[beta]40 (p=0.0002) and a 22% decrease in total brain A[beta]42 (p=0.0096; Table 1). At 6 months of age, scyllo-inositol treatment resulted in a 32% reduction in Aβ40 content (p=0.04) and a 20% reduction in Aβ40 content (p=0.02) compared to untreated TgCRND8 mice.

Since the decrease in Aβ concentrations detected after inositol treatment may be caused by altered Aβ extravasation into the plasma, Aβ-β levels in plasma were measured at 4 and 6 months of age (Table 1) . TgCRND8 mice had high plasma A[beta] concentrations at 4 months of age and remained constant at 6 months of age, even though CNS plaque burden still increased at 6 months of age (Table 1). Neither epi-inositol nor scyllo-inositol treatment had any effect on plasma A[beta] levels compared to untreated TgCRND8 mice (p=0.89). The most parsimonious explanation for this observation is that myo-inositol selectively alters A[beta] fibrosis in the CNS, but has no effect on [beta]- or [gamma]-secretase activity, or the normal mechanism of A[beta] clearance to plasma. However, this observation is significant for two reasons. First, in untreated AD patients, a decrease in Aβ levels in plasma and CSF is usually detected as the clinical course progresses (Mayeux et al., Ann. Neurol 46, 412, 2001). Second, AN1792 immunization study patients who developed strong antibody responses and overt clinical responses did not have altered plasma Aβ-β levels (Hock et al., Neuron 38, 547 2003). Therefore, these results indicate that changes in plasma A[beta] levels are not necessary for effective therapeutic outcomes.

To confirm that inositol stereoisomers have no effect on APP expression or proteolytic processing, the levels of APP holoprotein, sAPP-α, and various Aβ species were examined in the brains of inositol-treated and untreated TgCRND8 mice. Consistent with our previously reported data (McLaurin et al., Nat. Med. 8, 1263, 2002), Aβ42, Aβ40, and Aβ38 were the predominant species in the brains of TgCRND8 mice (Fig. 3A), and immature It was indistinguishable from the CNS content of mature glycolyzed APP (Fig. 3B) and that of sAPP-α. Taken together, these results suggest that epi-inositol and scyllo-inositol play a direct and selective role on Aβ oligomerization but not APP processing.

Changes in Aβ-β peptide burden were accompanied by a significant reduction in plaque burden (Table 1; Figures 2A-2I). Epi-inositol-treated TgCRND8 mice showed a significant decrease in mean plaque diameter compared to untreated TgCRND8 mice at 4 months of age but not 6 months of age (95 ± 4.3 μm ^vs 136 ± 15 μm ^{, respectively} , p=0.04; 370±9 μm ² vs. 423±22 μm ² , p=0.06). These results indicate that epi-inositol prevents A[beta] oligomerization at moderate A[beta] levels, but fails to inhibit microfibril formation once higher A[beta] concentrations occur. At 4 months of age, mean plaque diameter in the scyllo-inositol treatment group decreased from 136±15 μm ² to 103±4 μm ² (p=0.01). At 6 months of age, scyllo-inositol-treated TgCRND8 mice showed a reduction in Aβ peptide content, accompanied by a 20% reduction in the number of plaques (p=0.005) and a 35% reduction in the brain area covered by plaques (p=0.015) and mean plaque size decreased (339±10 μm ² vs. 423±21 μm ² , p=0.009). Across tests, these results confirmed a reduction in plaque burden following scyllo-inositol treatment.

Example 4 - Reduction of Glial Activity and Inflammation

Astroglial and microglial responses are neuropathological hallmarks of human AD and all amyloid mouse models (Irizarry et al., J Neuropathol Exp Neurol. 56, 965, 1997; K.D. Bornemann et al., Ann N Y Acad Sci. 908, 260, 2000). Therefore, the therapeutic effects of epi-inositol and scyllo-inositol on astrogliosis and microgliosis in the brains of TgCRND8 mice were investigated ( FIGS. 3A-3D ). Serial sagittal sections were stained with the astrocyte marker collagen fibrillary acidic protein (GFAP) and quantified as the percentage of brain area covered by astrogliosis. TgCRND8 mice had high basal astrogliosis (0.459 ± 0.048%) at 4 months of age, a slight increase (0.584 ± 0.089%) by 6 months of age, and was not limited to plaque area (Fig. 2A-2C). At 6 months of age, epi-inositol reduced the astroglial hyperplastic response to 0.388 ± 0.039% (p = 0.04; Fig. 2D-F). On the other hand, scyllo-inositol more effectively The qualitative proliferative response was reduced to 0.269±0.028% (p=0.006; Figure 2G-I). Scyllo-inositol-treated TgCRND8 mice also had significantly attenuated microglial activation (0.20±0.008% brain area). However, no significant reduction in microglial activation was demonstrated in epi-inositol treated mice at 6 months of age (0.248±0.02%; p=NS). Taken together, these data suggest that scyllo-inositol treatment reduces Aβ-induced inflammatory responses in the CNS.

Example 5 - Vascular Amyloid Burden

Alzheimer's disease is characterized by the presence of parenchyma and vascular amyloid deposits. In 6-month-old TgCRND8 mice, approximately 0.03% of the brain area was associated with vascular amyloid. In the 6-month-old epi-inositol-treated group, no difference in vascular amyloid burden was observed (Fig. 3C). In contrast, the vascular amyloid burden in the scyllo-inositol treatment group was significantly reduced (p=0.05) (Fig. 3C), and amyloid deposition was mainly confined to smaller blood vessels, less than 25 ^m in diameter (56±2% vs. 70±8% in small vessels of untreated TgCRND8 mice). The mean size of cerebrovascular plaques was significantly reduced in scyllo-inositol-treated mice compared to untreated mice (154±16 vs. 363±34, p=0.008; FIG. 3D ).

Example 6 - Improved Survival Rate

The survival rate of TgCRND8 mice was 50% at day 175, which increased to 72% after scyllo-inositol treatment (n=35 for each group, p<0.02 for scyllo-inositol vs. control group, FIG. 10B ). Treatment with myo-inositol did not significantly affect overall survival (Figure 10A). Controlled experiments confirmed that the increased survival of scyllo-inositol-treated mice was not an indirect effect of increased caloric intake. Thus, treatment of wild-type mice with scyllo-inositol had no effect on survival or other parameters such as body weight, coat condition or cage behavior. Furthermore, there were no differences in body weight, coat condition, or cage behavior between inositol-treated TgCRND8 mice and untreated TgCRND8 mice. Similar experiments were performed with mannose, a monosaccharide of similar molecular weight, which also had no effect on survival of TgCRND8 mice.

Example 7 - Treatment and Reversal of Amyloid Deposition

Taken together, prevention studies demonstrate that scyllo-inositol inhibits amyloid deposition in both parenchyma and blood vessels, thereby improving survival and cognitive function in a TgCRND8 mouse model of Alzheimer's disease. However, most Alzheimer's patients are likely to seek treatment only at the onset of symptoms and when Aβ oligomerization, deposition, toxicity and plaque formation have reached high levels in the CNS. Pilot experiments were therefore initiated with 5-month-old TgCRND8 mice. These mice had significant Aβ and plaque accumulation compared to human brains with AD.

Treatment Research Methods

Mice - Myo-inositol, epi-inositol and scyllo-inositol were fed to the TgCRND8 mice of the test group at an amount of 30 mg/mouse/day. One group entered the study starting at 5 months of age and the results were analyzed at 6 months of age. Body weight, coat characteristics, and cage behavior were monitored. All experiments were performed in accordance with the Canadian Council's Animal Care Guidelines.

Survival Survey - Survival probability is estimated by the Kaplan-Meier technique and is calculated and analyzed whenever a death occurs, making it suitable for small sample sizes. 35 mice per treatment group were used for survival analysis. Comparisons between treatment groups were documented using the Tarone-Ware test.

Behavioral Test - Reversal Study - Without pre-training, mice were entered into a Morris water maze test with a hidden platform. Mice were trained 6 times a day for 3 days. On the fourth day, the platform was removed from the pool and each mouse was subjected to a 30 second swim probe trial. On the last day, the animals were given a cue test to assess swimming ability, visual field and general cognition. In the cue trial, the platform was placed in a signal area different from that used for the test and marked with a flag. Allow the animal 60 seconds to find the platform. Animals that did not find the platform were not used in the final analysis of spatial memory. Behavioral data were analyzed using a mixed model of factorial analysis of variance (ANOVA) with drug or genotype and training period as repeated measures factors.

Brain Amyloid Burden - Brains were removed and one hemisphere was fixed in 4% paraformaldehyde and embedded in paraffin along the mid saggital plane. To generate a systematic set of uniform random sections, the entire hemisphere was transected to collect 5 μm serial sections. Groups of sections separated by 50 mm were used for analysis (10-14 slices/group). Plaques were identified after antigen retrieval with formic acid and incubated with primary anti-Aβ antibody (Dako M-0872) followed by secondary antigen (Dako StreptABCcomplex/horseradish kit). The final product was visualized by DAB counterstaining with hematoxylin. The burden of amyloid plaques was evaluated using Leco IA-3001 image analysis software connected to a Leica microscope and a Hitachi KIP-MU CCD camera.

Plasma and Brain Aβ Content - Brain hemisphere samples were homogenized in sucrose buffer, then either 0.4% diethylamine/100mM NaCl was added to solubilize Aβ content, or cold formic acid was added to separate total Aβ. After neutralization, samples were diluted and analyzed for Aβ40 and Aβ42 using commercially available kits (BIOSOURCE International). Each hemisphere was analyzed three times, and the mean ± standard error of the mean was reported.

Results and Significance - All animals participating in the reversal study survived and showed no outward signs of distress or intoxication. Cognitive function in TgCRND8 mice was assessed using a 3-day experimental paradigm using the spatially referenced memory model of the Morris water maze (Figures 4-8). A mixed model of analysis of variance (ANOVA) was used to determine Data obtained from treated and untreated TgCRND8 mice as well as data from treated and untreated non-Tg littermates were analyzed (n=10 for all combinations). Compared with wild-type littermates, TgCRND8 mice were significantly less competent (Fig. 4). In contrast, there was no difference between scyllo-inositol-treated TgCRND8 mice and non-Tg littermates on all days (p=0.38; Figure 5). Epi-inositol treated TgCRND8 mice were almost significantly different compared to treated non-Tg littermates (p=0.07; Figure 6). Similarly, myo-inositol treated TgCRND8 mice were also significantly different from treated non-Tg littermates (p=0.05; Figure 7). When comparing the Riess water maze test learning period between treatment groups, all mice behaved similarly (Figure 8). In contrast, there was no difference between the scyllo-inositol group and non-Tg sibling mice (Fig. 8). Thus, in fact, scyllo-inositol reversed cognitive deficits to such an extent that there was no difference between scyllo-inositol-treated TgCRND8 mice and normal mice. This improved performance was not due to nonspecific effects on behavioral, motor, or sensory systems, as epi-inositol and scyllo-inositol treatments had no effect on performance in non-Tg mice. This improved performance was also not due to nutritional or caloric effects, as there were no differences in body weight, activity, and coat condition between the treated and untreated groups.

To determine whether improved cognition was associated with reduced plaque accumulation and Aβ burden, brain tissue was autopsied. Changes in cognition were accompanied by corresponding changes in plaque stock and Aβ load (Figure 9 and Table 2). Myo-inositol treatment had no effect on plaque accumulation or A[beta] burden (Figure 9 and Table 2). Epi-inositol-treated TgCRND8 mice had no significant reduction in mean plaque diameter compared to untreated TgCRND8 mice (Fig. 9), but Aβ burden was significantly reduced (Table 2). These results suggest that epi-inositol prevents Aβ oligomerization at moderate Aβ levels, but fails to completely inhibit microfibril formation once higher Aβ concentrations occur. Plaque accumulation and Aβ load were significantly reduced in the scyllo-inositol treatment group. Across tests, these results confirmed a reduction in plaque accumulation following scyllo-inositol treatment. These results are comparable in size to the 6-month prophylaxis study and further support the potential of scyllo-inositol.

Since the decrease in A[beta] concentrations detected after inositol treatment may be due to altered A[beta] extravasation into plasma, we measured A[beta] levels in plasma (Table 2). TgCRND8 mice had high plasma Aβ concentrations at 6 months of age. Neither myo-inositol, epi-inositol, or scyllo-inositol treatment had any effect on plasma A[beta] levels compared to untreated TgCRND8 mice (p=0.89). The most parsimonious explanation for this observation is that myo-inositol selectively alters A[beta] fibrosis in the CNS, but has no effect on [beta]- or [gamma]-secretase activity, or the normal mechanism by which A[beta] is cleared into plasma. However, this observation is significant for two reasons. First, in untreated AD patients, decreased Aβ levels in plasma and CSF are routinely detected as the progression of the clinical course. Second, there were no altered plasma A[beta] levels in the AN1792 immunization study patients who developed strong antibody responses and overt clinical responses. Therefore, these results further suggest that alteration of plasma A[beta] levels is not necessary for effective therapeutic outcomes.

Taken together, these data reveal that in a transgenic mouse model of Alzheimer's disease, in the "late presymptomatic" stage, before mice develop overt cognitive deficits and amyloid neuropathology, select Scyllo-inositol can eliminate Aβ accumulation in the brain, brain amyloid plaque deposition and cognitive decline. Furthermore, these compounds were effective in reversing amyloid deposition, neuropathology, and cognitive deficits even when scyllo-inositol was administered after cognitive deficits and amyloid plaque neuropathology. Thus, these results indicate that scyllo-inositol is effective in preventing disease and treating disease in patients diagnosed with AD.

Example 8 - Two-month treatment study of scyllo-inositol

To determine the longer-term extent of scyllo-inositol treatment of disease, 5-month-old TgCRND8 mice were fed scyllo-inositol or not treated for 2 months (n=10 per group). Seven-month-old scyllo-inositol-treated TgCRND8 mice were compared with untreated TgCRND8 mice and treated non-Tg littermates using a three-day Morris water maze test. Behavioral data were analyzed using a mixed model of factorial analysis of variance (ANOVA) with drug and genotype as between-subject variables and training period as a within-subject variable. As seen with scyllo-inositol treatment for 1 month (Figure 12A), there was no difference between scyllo-inositol-treated TgCRND8 mice for 2 months and non-Tg littermates treated with scyllo-inositol (Figure 12B). To link improved cognition to pathology, Aβ40 and Aβ42 levels in the brain were analyzed (Table 3). Both insoluble Aβ40 and Aβ42 levels decreased by 20% after scyllo-inositol treatment. These results demonstrate that scyllo-inositol acts continuously during disease development.

Table 3 Inositol treatment reduces the content of Aβ40 and Aβ42

ANOVA using Fisher's PLSD, ^* p<0.05.

Example 9 - Effect of dosage on pathological outcome of TgCRND8 mice with disease

5-month-old TgCRND8 mice were fed once a day with scyllo-inositol dissolved in water at doses of 10 mg/Kg, 30 mg/Kg and 100 mg/Kg or without treatment. Animals were sacrificed one month after treatment and pathological results were analyzed. Analysis of Aβ levels in the brains of all groups confirmed that all drug doses were equally effective in reducing soluble Aβ42 levels compared to untreated TgCRND8 mice (20% decrease, F _3,15 =3.1, p=0.07; Figure 13A). Individual dose analysis demonstrated that the 10 mg/Kg and 30 mg/Kg dose groups were significantly different from the untreated control group (p=0.03 and p=0.02, respectively). The selected doses were not significantly different from each other (F _2,11 =0.6, p=0.57; FIG. 13A ). Feeding dose was significantly different for insoluble Aβ42 (F _3,15 =0.69, p=0.58; FIG. 13B ) or soluble and insoluble Aβ40 (F _3,15 =0.04, p=0.99 and F _3,15 = 0.36, p=0.79; Figures 14A and 14B) No significant effect.

Example 10 - Therapeutic effect of iso-inositol on diseased TgCRND8 mice

To assess whether iso-inositol could also be effective in preventing further development and/or partially reversing an established AD-like phenotype, initiation of treatment of TgCRND8 mice was delayed until 5 months of age. Groups of TgCRND8 mice and non-transgenic littermates were either treated with iso-inositol for 28 days or not. In these trials, compound dosing and oral administration, as well as behavioral and neurochemical assessments were performed in the same manner as in the treatment trials described above.

The 6 month old group of iso-inositol treated TgCRND8 mice performed significantly better than untreated TgCRND8 mice (F _1,13 =0.45, p=0.05; data not shown). The cognitive performance of 6-month-old iso-inositol-treated TgCRND8 mice was also significantly different from that of non-transgenic littermates. This beneficial effect of inositol treatment was not due to non-specific effects on behavioral, motor or sensory systems, as inositol treatment had no effect on cognitive performance in non-Tg mice (F _1,12 = 0.98; p = 0.49). Brain Aβ content of treated versus untreated TgCRND8 mice was analyzed to determine whether improved behavior was associated with altered Aβ (Table 4). Iso-inositol treatment reduced soluble Aβ42 (20% decrease, p<0.05), an effect similar to that observed for scyllo-inositol. Iso-inositol did not significantly alter insoluble A[beta]42 or A[beta]40 (both soluble and insoluble pools). One possible explanation for the decrease in Aβ42 is the clearance of peripheral Aβ42, which subsequently leads to an increase in plasma Aβ42. The plasma Aβ42 content after iso-inositol treatment was not different from the plasma content of untreated TgCRND8 (Table 5). Consistent with other inositol stereoisomers, these results demonstrate that plasma A[beta] levels are not affected by iso-inositol treatment.

Table 4. Iso-inositol treatment reduces Aβ42 levels

Analysis of variance using Fisher's PLSD, ^* p<0.05.

Table 5 Blood Biochemistry - Scyllo-inositol Dose Study

Example 11 - Inositol treatment does not affect blood chemistry

To exclude any deleterious effect of inositol treatment on blood chemistry and organ function, blood was analyzed after 1 month of scyllo-inositol and iso-inositol treatment (Tables 5 and 6). There were no significant differences in total protein, albumin, globulin, bilirubin, alkaline phosphatase, glucose, urea, and creatinine between the treated groups or from untreated TgCRND8 mice. All levels fell within normal ranges determined by non-transgenic wild-type mice. In addition to hemolysis, jaundice and lipemia were also normal. These results suggest that iso-inositol and scyllo-inositol exhibit no deleterious effects on blood chemistry and organ function.

Table 6 Blood Biochemistry - 1 Month Treatment Study

Example 12 - Double transgenic mouse model of scyllo-inositol on Alzheimer's disease Role of PS1×APP in preventing AD-like pathology

Tg PS1×APP mice are an enhanced model of Alzheimer's disease expressing a mutant human PS1 transgene encoding two familial mutations (M146L and L286V) and a human APP transgene encoding the Indiana and Swedish familial mutations . After 30-45 days of age, these animals developed robust expression of brain Aβ levels and amyloid deposition. In a prophylactic trial, TgPSlxAPP mice were treated with scyllo-inositol from weaning and neuropathological effects were evaluated at 2 months of age (Figures 16 and 17). Scyllo-inositol-treated TgPS1×APP mice showed a significant reduction in all tests of plaque burden at 2 months of age compared to untreated TgPS1×APP mice (% brain area covered by plaque = 0.157 ±0.007 vs=.065±0.016, p<0.001; mean plaque size=177±8 ^μm2 vs 149±5 ^μm2 , p<0.05; plaque count 3054±324 vs 1514±510, p<0.01; Figure 17) . These results demonstrate that scyllo-inositol prevents amyloid deposition in two robust models of Alzheimer's disease.

Example 13 - Effect of increased caloric intake on TgCRND8 mice

To rule out the contribution of increased caloric intake or nonspecific effects, TgCRND8 mice were treated with the monosaccharide mannitol of similar molecular weight. At 6 months of age, mannitol-treated TgCRND8 mice were indistinguishable from untreated TgCRND8 mice (Fig. 11A) and significantly different from mannitol-treated non-Tg littermates (Fig. 11B). . Mannitol had no effect on the behavior of non-Tg mice because mannitol-treated non-Tg littermates did not differ from untreated non-Tg mice. These results in relation to pathological studies indicated that mannitol did not alter plaque burden in TgCRND8 mice (Fig. 11C). At the same time, the detection of the survival rate proved that mannitol had no effect on the survival rate of TgCRND8 mice ( FIG. 11D ).

While the invention has been described in terms of specific embodiments thereof, it is apparent to those skilled in the art that numerous other changes and modifications, as well as other uses. Accordingly, the invention is not to be limited by what has been specifically disclosed herein, but only by the appended claims.

Claims (5)

1. Use of scyllo-inositol in the preparation of a reagent used in a method for diagnosing abnormally folded or accumulated proteins and/or amyloid fibrils or amyloid in a subject, wherein the method comprises the steps of: (a) administering to said subject radioactive scyllo-inositol or labeled with a sufficient amount and emitting under conditions that allow said scyllo-inositol to bind to abnormally folded or accumulated proteins and/or fibrils or amyloid scyllo-inositol as a signal detectable substance if abnormally folded or accumulated proteins and/or fibrils or amyloid are present; and (b) detecting a signal radioactive or detectable signal from scyllo-inositol bound to abnormally folded or accumulated protein and/or fibrils or amyloid, thereby diagnosing the abnormally folded or accumulated protein in said subject and/or the presence of amyloid fibrils or amyloid. 2. The use of claim 1, wherein the detectable signal is an enzyme-linked immunosorbent assay signal. 3. The use of claim 1, wherein the detectable signal is a fluorescent signal. 4. The use of claim 1, wherein the detectable signal is a radioactive signal. 5. The use of claim 1 , 2, 3 or 4, wherein the reagent is used to diagnose Alzheimer's disease.

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