WO2011047374A1

WO2011047374A1 - Methods of treating tauopathies

Info

Publication number: WO2011047374A1
Application number: PCT/US2010/053041
Authority: WO
Inventors: Giulio Pasinetti; Jun Wang
Original assignee: Mount Sinai School Of Medicine Of New York University
Priority date: 2009-10-18
Filing date: 2010-10-18
Publication date: 2011-04-21

Abstract

The disclosure provides methods for preventing or treating tauopathies by administering a therapeutically effective amount of demebolin, or generally a hydrogenated pyrido[4,3-b]indole derivative. Also provided are methods for ameliorating a symptom associated with a tauopathy.

Description

METHODS OF TREATING TAUOPATHIES

Field

The disclosure relates to the therapeutic use of antihistamine compounds to treat, prevent or ameliorate neurodegenerative disease processes.

Background

Neurodegenerative diseases form a substantial class of diseases in humans that annually exact a significant toll in terms of physical and mental anguish and loss as well as health dollars spent. Over the past few decades, an increasing amount of information has been obtained to further our understanding of these disease processes, on occasion down to the molecular level. Much remains to be learned, however. One protein that appears to be involved in a number of neurodegenerative diseases is the Tau protein.

Tau proteins are microtubule-associated proteins that are abundant in neurons in the central nervous system and are less common elsewhere. Tau proteins interact with tubulin to stabilize microtubules and promote tubulin assembly into microtubules. Tau has two ways of controlling microtubule stability: isoforms and phosphorylation. Six tau isoforms exist in brain tissue, and they are distinguished by the number of binding domains. Three isoforms have three binding domains and the other three have four binding domains. The binding domains are located in the carboxy-terminus of the protein and are positively-charged (allowing binding to the negatively-charged microtubule). The isoforms with four binding domains confer greater stability on microtubules than those with three binding domains. The isoforms are a result of alternative splicing in exons 2,3, and 10 of the tau gene.

Phosphorylation of tau is regulated by a host of kinases. For example, PKN, is a serine/threonine kinase that, when activated, phosphorylates tau, resulting in disruption of microtubule organization. Hyperphosphorylation of the tau protein (tau inclusions), however, can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the pathogenesis of Alzheimer's disease.

Tau protein is a highly soluble microtubule-associated protein (MAP). In humans, these proteins are mostly found in neurons. One of tau's main functions is to modulate the stability of axonal microtubules. Tau is not present in dendrites and is active primarily in the distal portions of axons where it provides microtubule stabilization but also flexibility, as needed. The tau gene is located on chromosome 17q21 and contains 16 exons. The major tau protein in the human brain is encoded by 11 exons. Exons 2, 3 and 10 are alternatively spliced, allowing six combinations. Thus, in the human brain, the tau proteins constitute a family of six isoforms with a range from 352-441 amino acids. The isoforms differ in having 0-2 inserts of 29 amino acids at the N-terminal region (exon 2 and 3), and three or four repeat- regions at the C-terminal region of exon 10 may be lacking. Thus, the longest isoform in the CNS has four repeats (Rl, R2, R3 and R4) and two inserts (441 amino acids total), while the shortest isoform has three repeats (Rl, R3 and R4) and no insert (352 amino acids total). All six tau isoforms are present in an often hyperphosphorylated state in paired helical filaments in brains of patients with Alzheimer's disease. When misfolded this otherwise very soluble protein can form extremely insoluble aggregates that contribute to a number of

neurodegenerative diseases.

Dimebolin, also known as latrepirdine, is an antihistamine drug with the formula of 2,3,4,5-Tetrahydro-2,8-dimethyl-5-(2-(6-methyl-3-pyridyl)ethyl)-lH-pyrido(4,3-b)indole. A commercially available hydrochloride salt is Dimebon. Dimebolin is an orally active small molecule that has been shown to inhibit brain cell death in preclinical studies of Alzheimer's disease and Huntington's disease, making it a potential treatment for these diseases. Research indicates that it may also have cognition-enhancing effects in healthy individuals, in the absence of neurodegenerative disease pathology. Recently dimebolin has attracted renewed interest after being shown to have positive effects on persons suffering from Alzheimer's disease. Animal studies show that dimebolin has potential beneficial effects on Alzheimer's disease models. Preliminary results from an initial six-month phase II human trial have shown that at 12 months, there was significant improvement over placebo. Dimebolin appears to operate through multiple mechanisms of action, both blocking the action of neurotoxic beta-amyloid proteins and inhibiting L-type calcium channels, modulating the action of AMP A and NMD A glutamate receptors, and it may exert a neuroprotective effect by blocking a novel target that involves mitochondrial pores, which are believed to play a role in the cell death that is associated with neurodegenerative diseases and the aging process. Dimebolin also blocks a number of other receptors including a- Adrenergic receptors and the serotonin receptor subtypes 5-HT2C, 5-HT5A and 5-HT6. Thus, despite its promise as an Alzheimer's disease and Huntington's disease therapeutic, the multiple mechanisms of action and lack of detailed knowledge concerning the activities of dimetabolin have confined its recognized usefulness to the treatment of these diseases.

For the reasons elaborated above, it is apparent that a need continues to exist in the art for therapeutics useful in treating neurodegenerative disease processes. Summary

The technology disclosed herein satisfies at least one of the aforementioned needs in the art in providing methods for treating a range of neurodegenerative disease conditions with a therapeutically effective amount of dimebolin.

The disclosure provides a method for treating a tauopathy comprising administering to a patient in need a therapeutically effective amount of dimebolin or a pharmaceutically acceptable salt thereof . Any pharmaceutically acceptable salt known in the art is contemplated. In addition, the disclosure contemplates the use of any hydrogenated pyrido[4,3-b]indole derivative in the methods according to the disclosure. In some embodiments, the tauopathy is selected from the group consisting of diseases exhibiting predominant tau pathology and prominent parkinsonism, diseases exhibiting predominant tau pathology and variable parkinsonism, diseases exhibiting predominant tau pathology and uncommon parkinsonism and diseases exhibiting tau pathology associated with amyloid deposition, with the exception of Alzheimer's disease. Exemplary diseases exhibiting predominant tau pathology and prominent parkinsonism are selected from the group consisting of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), postencephalitic parkinsonism (encephalitis lethargica), parkinsonism-dementia complex of Guam (Kii, Papua) and Guadeloupean parkinsonism. Exemplary diseases exhibiting predominant tau pathology and variable parkinsonism are selected from the group consisting of frontotemporal dementias with Parkinsonism linked to chromosome 17 (FTDP-17).

Exemplary diseases exhibiting predominant tau pathology and uncommon parkinsonism are selected from the group consisting of argyrophilic grain disease (AgD) and Pick's disease (PiD). Exemplary diseases exhibiting tau pathology associated with amyloid deposition are selected from the group consisting of dementia pugilistica (prominent parkinsonism), Alzheimer's disease (parkinsonism variable), Down syndrome, Familial British

dementia/familial Danish dementia. Exemplary diseases exhibiting Tau deposition with other pathology are selected from the group consisting of Niemann-Pick disease type C, neurodegeneration with brain iron accumulation type 1, subacute sclerosing panencephalitis (SSPE) and Myotonic dystrophy.

Other features and advantages of the present disclosure will be better understood by reference to the following brief description of the drawing and detailed description. Brief Description of the Drawing

Figure 1 provides data showing that dimebolin improves cognitive function without affecting amyloid beta content.

Figure 2 shows that chronic dimebolin treatment does not affect brain or plasma levels of Αβ peptide.

Figure 3 discloses experimental results showing that dimebolin does not affect APP protein processing.

Figure 4 provides results of experiments that establish that dimebolin does not improve mitochondria function in the brain.

Figure 5 establishes that dimebolin treatment reduces both maximum respiratory capacity and extracellular acidification.

Figure 6 shows that dimebolin is well-tolerated by mice.

Figure 7 provides data showing that dimebolin treatment significantly reduces tau protein phosphorylation.

Figure 8 provides data showing the effect of Dimoblin on Akt and MAP kinase signaling in primary neurons derived from E15 wild type mice. (A) Time course of Akt, Erk, p70 S6 and JNK phosphorylation in primary neurons treated with 200nM Dimoblin for 15, 30 and 60m minutes. Phospho-Akt was measured by western blot using p437-Akt antibody. The multiplex signaling kit from Millipore was used to determine the phospho- Erk/MAPK (Thrl85/Tyrl87), JNK (Thrl83/Tyrl85), and p70 S6 (Thr412). (B) Time course of tau phosphorylation at serl99/ser202/thr205 upon Dimoblin treatment.

Detailed Description

Tau, a microtubule-associated protein (MAP), is the main constituent of neurofibrillary tangles (NFTs). The MAP tau is as an elongated molecule (about 35-50 nm long, dependent on the isoform) without recognizable secondary structure. Tau's role may be likened to that of ties that hold the microtubular tracks in place. The finding that mutations in the tau gene are responsible for frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has established that tau protein plays a key role in neurodegeneration. These observations are consistent with distinct sets of tau isoforms being expressed in different neuronal populations leading to different pathologies. Tau is enriched in axons, probably playing a role in axonal development, and it is the inappropriate hyperphosphorylation of this protein in Alzheimer' s disease that contributes to neurofibrillary tangle development. The amino acid sequence of the Tau protein is provided in SEQ ID NO:2 and in Table 1. The sequence of MAPT, the gene encoding Tau, is provided in SEQ ID NO:l and in Table 1.

Table 1

A tauopathy, as defined herein, is a member of a class of neurodegenerative diseases, other than Alzheimer's disease and Huntington's disease, resulting from the pathological aggregation of tau protein in so-called tangles in the human brain. Examples of tauopathies as defined herein are: frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, and frontotemporal lobar degeneration, also known as Pick's disease. Also contemplated by the disclosure as a tauopathy is Parkinson' s disease and amyotrophic lateral sclerosis (ALS).

Tauopathies with parkinsonism represent a spectrum of disease entities unified by the pathologic accumulation of hyperphosphorylated tau protein fragments within the central nervous system. The term tauopathies refers generally to neurodegenerative diseases with prominent tau pathology in the CNS, predominantly within the neuronal compartment, but also within glial cells. Tau is an abundant microtubule-associated protein, physiologically expressed in neurons. In tauopathies, the soluble tau protein detaches from microtubules and forms abnormal, fibrillar structures of aggregated, hyperphosphorylated, and ubiquinated tau. The molecular composition of tau aggregates in tauopathies is becoming better understood, resulting in the definition of etiologically heterogeneous, clinically and neuropathologically overlapping disease entities. Some tauopathies are characterized by parkinsonism, which may be partially responsive to levodopa; others are characterized by dementia with signs of frontal lobe dysfunction; still others are characterized by a motor neuron disorder phenotype.

The tauopathies amenable to the methods disclosed herein can be organized by consideration of the neuropathology and clinical syndromes, resulting in the following categories: 1) Predominant tau pathology/prominent parkinsonism, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Postencephalitic parkinsonism (encephalitis lethargica), Parkinsonism-dementia complex of Guam (Kii, Papua),

Guadeloupean parkinsonism, Miscellaneous cases -'tangle parkinsonism,'LRRK2 mutations; 2) Predominant tau pathology/variable parkinsonism, including frontotemporal dementias with Parkinsonism linked to chromosome 17 (FTDP-17); 3) Predominant tau

pathology/parkinsonism uncommon, including argyrophilic grain disease (AgD) and Pick's disease (PiD); 4) Tau pathology associated with amyloid deposition, including Dementia pugilistica (prominent parkinsonism), Alzheimer' s disease (parkinsonism variable), Down syndrome, Familial British dementia/familial Danish dementia; and 5) Tau deposition with other pathology, including Niemann-Pick disease type C, neurodegeneration with brain iron accumulation type 1 , subacute sclerosing panencephalitis (SSPE) and Myotonic dystrophy.

Consistent with the foregoing discussion, tauopathies are characterized by the progressive, age-dependent intracellular formation of misfolded tau protein aggregates that play key roles in the initiation and progression of neuropathogenesis. Disclosed herein is recent work that led to the identification of dimebolin (CAS number 3613-73-8) as a chemical compound useful for treating and/or preventing misfolded-tau protein-mediated neurodegenerative disorders. In vitro studies demonstrated that dimebolin attenuated tau protein-mediated neuropathologic mechanisms by preventing abnormal tau phosphorylation. Hyperphosphorylated Tau protein is the main component of paired helical filaments (PHF) and PHF is the main component of neurofibrillary tangles, the pathological hallmark of Alzheimer's disease (AD). Moreover, preclinical feasibility evidence indicated that dimebolin treatment significantly reduced the level of phosphorylated tau protein content in the brain without alteration of total tau protein expression. This reduction was accompanied by a significantly reduced level of PHF and tangles in both the hippocampus and cerebral cortex. These studies establish that dimebolin is an effective agent for treating tau-mediated neurodegenerative disorders.

Disclosed herein are data establishing the unexpected finding that dimebolin is useful in the prevention, treatment and/or amelioration of tauopathies. Although not wishing to be bound by theory, it is believed that dimebolin may exert its therapeutic effect by influencing the formation or retention of tau tangles. Additionally, it is expected that derivatives of dimebolin, generally envisaged as hydrogenated pyrido[4,3-b]indole derivatives, will be useful in preventing, treating or ameliorating tauopathies. Within this group of compounds, diazoline (mebhydroline), dimebon, dorastine, carbidine(dicarbine), stobadine, and hevotroline, based on tetra- and hexahydro-lH-pyrido[4,3-b]indole derivatives, are commercially available. Exemplary compounds include 2-methyl-2,3,4,5-tetrahydro-lH- pyrido[4,3-b]indole; 2,8-dimethyl-2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indole and its methyliodide; cis(+-)2,8-dimethyl-2,3,4,4a,5,9b-hexahydro-lH-pyrido[4,3-b]indole and its dihydrochloride salt; 2-methyl-8-bromo-2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indole and its hydrochloride salt; 2-ethyl-2,3,4,5-tetrahydro-lH-pyrido[4,3-b]indole; 2-benzyl-2,3,4,5- tetrahydro-lH-pyrido[4,3-b]indole; 2,8-dimethyl-5-benzyl-2,3,4,5-tetrahydro-lH-pyrido[4,3- b]indole and its hydrochloride salt; 2-methyl-5-[2-(6-methyl-3-pyridyl)ethyl]-2,3,4,5- tetrahydro-lH-pyrido[4,3- -b]indole and the sesquisulfate monohydrate thereof; and 2,8- dimethyl-5-[2-(6-methyl-3-pyridyl)ethyl]-2,3,4,5-tetrahydro-lH-pyrido- [4,3-b]indole and its dihydrochloride salt.

Dimebon and its derivatives may be administered by any known route and it may be formulated with buffers, excipients, and other known compounds into a variety of physiologically compatible pharmaceutical compositions. In one form, dimebon is provided in the form of tablets (comprising 10 mg of dimebon, 30 mg of lactose, and 5 mg of magnesium stearate) for oral administration in a dose of 0.02 g three times per day. The technology disclosed herein is illustrated by the following examples, wherein Example 1 shows that dimebolin improves cognitive function; Example 2 demonstrates that dimebolin does not affect brain or plasma levels of Αβ peptide; Example 3 provides data that establishes that dimebolin does not affect APP processing; Example 4 shows that dimebolin does not improve mitochondrial function; Example 5 illustrates the effects of dimebolin in reducing respiratory capacity and extracelllular acidification; Example 6 provides data showing that dimebolin is well-tolerated by mammals; and Example 7 shows that dimebolin reduces phosphorylation of the Tau protein.

Example 1

The TgCRND8 mouse model of Alzheimer's disease (AD) was used to assess the cognitive effects of chronic dimebolin administration. TgCRND8 mice were divided into treated and untreated groups, with the treated group receiving daily dosing of dimebolin over the indicated time. All mice were subjected to the Morris water maze test in which an animal is placed in a chamber containing water and a submerged platform in one quadrant. The animals are forced to swim unless they have located the platform upon which they can stand. The influence of chronic dimebolin treatment on Αβ-related spatial memory in TgCRND8 mice vs. untreated control mice is shown in Figure 1. (A) Hidden platform acquisition (left panel) latency score represents the time taken to escape to the platform from the water). (B) Probe trial percent of time in quadrant is calculated as the ratio of time spent in the target quadrant area relative to the time spent in the rest of the pool. (C) Platform crossing is calculated as the times the animals cross the target platform. *, p < 0.05 (2-tailed student t- test ). (D) Visible trial and (E) average swimming speed analysis excludes the possibility that dimebolin treatment might affect non-spatial parameters, such as sensorimotor performance and motivation. The results establish that dimebolin improves cognitive function without affecting beta amyloid content in the mouse brain.

Example 2

The results described in Example 1 led to an investigation of whether the cognitive improvement attributable to dimetabolin treatment was due to altered levels of beta amyloid proteins in the brain. To measure beta amyloid protein levels as a function of dimetabolin treatment, TgCRND8 mice weree divided into two groups, i.e., a group receiving chronic dosing of metabolin and a control group not exposed to metabolin. Following the period of chronic exposure to dimetabolin, brain and blood samples of treated and untreated mice were obtained and assayed for Αβ1-40 and Αβ1-42 expression levels. The results are provided in Figure 2, wherein (A) provides the total brain Αβ1-40 and Αβ1-42 content in TgCRND8 mice, and (B) provides the plasma levels of Αβ1-40 and Αβ1-42.

Example 3

A study was conducted to assess whether dimebolin would affect the processing of the amyloid protein precursor (APP) in neuronal tissue. A set of in vitro cortical-hippocampal neuron cultures was established and one culture was exposed to dimebolin while another culture served as a negative control. Results of treating cortical-hippocampal neuron cultures are shown in Figure 3, i.e., amyloid peptide (A) Αβ1-40 and (B) Αβ1-42 in the conditioned medium of primary neuron culture derived from E16 of Tg2576 mice treated with varying doses of dimebolin for 24 hours. Apparent from the data in Figure 3, dimebolin has no effect on Αβ1-40 and Αβ1-42 concentrations in conditioned medium, and thus demebolin does not affect APP protein processing in primary neuron cultures.

Example 4

To determine if dimebolin was affecting brain biochemistry in a manner that led to a nonspecific benefit in apparent cognitive ability, e.g., through increased locomotion, the effect of dimebolin on brain mitochondrial enzymes was investigated. Brain tissue from TgCRND8 mice chronically exposed to dimebolin or not exposed to dimebolin were obtained using conventional techniques and assays of mitochondrial tricarboxylic acid (TCA) cycle enzyme activities were performed. The results are shown in Figure 4, wherein (A) shows that dimebolin has no effect on the citrate synthase activity of mitochondria isolated from the brains of TgCRND8 mice, (B) dimebolin treatment reduces the activity of malate dehydrogenase, and (C) dimebolin treatment has no effect on alpha-ketogluatarate dehydrogenase activity. Thus, dimetabolin is not yielding cognitive improvement by a general effect on brain biochemistry.

Example 5

Consistent with the results described in Example 4, experiments show that dimebolin treatment reduces both maximum respiratory capacity and extracellular acidification in primary neuron cultures. In vitro culture of E14 primary neurons showed that dimebolin treatment reduced both maximum respiratory capacity and extracellular acidification in these cells. The results are provided in Figure 5, wherein (A) shows the oxygen consumption rate in primary neuron culture treated with various doses of dimebolin for 24 hours, and (B) shows the maximum respiratory capacity in primary neuron culture treated with various concentrations of dimebolin for 24 hours. Figure 5(C) shows the FCCP-induced extracellular acidification rate in primary neuron culture treated with various concentrations of dimebolin for 24 hours.

Example 6

Therapeutic compounds approved for use in humans must be both effective and safe. To assess the safety profile of dimebolin, mice receiving dimebolin were examined for general toxic effects by measuring body weight and food consumption. The hTAU441V337M / R406W mouse model of tauopathy was used to assess the safety or dimebolin. Mice were divided into experimental and control groups and the experimental group received 10 mg/kg/day chronic doses of dimebolin. Measurement of body weight and food consumption showed that dimebolin administrations were well-tolerated by the mice. The results are presented in Figure 6 which shows (A) body weight and (B) food consumption as functions of time on dimebolin dosage.

Example 7

The effect of dimebolin on Tau protein chemistry in the brain was also examined, with surprising results. The study showed that chronic dimebolin treatment significantly reduced tau protein phosphorylation in hTau mice. Western blot analyses of brain proteins in hTau mice chronically exposed to dimebolin showed that chronic dimebolin treatment does not affect the level of total tau protein (inset) (Figure 7(A)), as detected using conventional blotting techniques and the Tau5 antibody. In contrast to the absence of any effect on Tau protein expression levels, dimebolin did reduce the level of Tau phosphorylation at amino acids 396 and 404 (inset; see also Table 1 and SEQ ID NO:2), as revealed by conventional Western blot analyses using the AD2 antibody to measure levels of phosphorylated Tau.

Example 8

Pathologic hyperphosphorylation and aggregation of tau play a central role in

neurodegeneration and neuronal dysfunction in AD as well as in other taoupathies. Dimoblin was thus tested with respect to the alteration of activities of proteins responsible for modifying tau phosphorylation. Primary neuron culture derived from E15 wild type mice was treated with 200nM of Dimoblin and investigated Akt-mediated and mitogen- activated protein (MAP)-mediated signaling cascades implicated in tau phosphorylation.

Encouragingly, it was observed that Dimoblin treatment activates Akt kinase by promoting phosphorylation at residue serine 473 without affecting the level of total Akt protein (Figure 8A). Activation of Akt may down regulate the activity of GSK-3P and contribute to inhibition of tau phosphorylation. We also found that treatment with Dimoblin resulted in a significant reduction in phosphorylation of MAPK (Erk), p70 S6, the c-Jun-N-terminal kinase (JNK) as detected using Luminex 200 multiplex based cell signaling kit (Millipore Inc.) (Figure3A). MAPK, JNK and p70S6 have been reported to phosphorylated tau protein. These changes were accompanied by reduced tau phosphorylation as probed by AT8, an antibody that specifically recognizes serl99/ser202/thr205 phosphorylation of tau (Figure 8B).

Numerous modifications and variations of the technology disclosed herein are possible in view of the above teachings and are within the scope of the invention. The entire disclosures of all publications cited herein are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a tauopathy comprising administering to a patient in need a therapeutically effective amount of a hydrogenated pyrido[4,3-b]indole derivative or a pharmaceutically acceptable salt thereof .

2. A method for treating a tauopathy comprising administering to a patient in need a therapeutically effective amount of dimebolin or a pharmaceutically acceptable salt thereof .

3. The method according to claim 2 wherein the tauopathy is selected from the group consisting of diseases exhibiting predominant tau pathology and prominent parkinsonism, diseases exhibiting predominant tau pathology and variable parkinsonism, diseases exhibiting predominant tau pathology and uncommon parkinsonism and diseases exhibiting tau pathology associated with amyloid deposition, with the exception of

Alzheimer's disease.

4. The method according to claim 3 wherein the diseases exhibiting predominant tau pathology and prominent parkinsonism are selected from the group consisting of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), postencephalitic parkinsonism (encephalitis lethargica), parkinsonism-dementia complex of Guam (Kii, Papua) and Guadeloupean parkinsonism.

5. The method according to claim 3 wherein the diseases exhibiting predominant tau pathology and variable parkinsonism are selected from the group consisting of frontotemporal dementias with Parkinsonism linked to chromosome 17 (FTDP-17).

6. The method according to claim 3 wherein the diseases exhibiting predominant tau pathology and uncommon parkinsonism are selected from the group consisting of argyrophilic grain disease (AgD) and Pick's disease (PiD).

7. The method according to claim 3 wherein the diseases exhibiting tau pathology associated with amyloid deposition are selected from the group consisting of dementia pugilistica (prominent parkinsonism), Alzheimer's disease (parkinsonism variable), Down syndrome, Familial British dementia/familial Danish dementia.

8. The method according to claim 3 wherein the diseases exhibiting Tau deposition with other pathology are selected from the group consisting of Niemann-Pick disease type C, neurodegeneration with brain iron accumulation type 1, subacute sclerosing panencephalitis (SSPE) and Myotonic dystrophy.