COMPOUNDS FOR TREATING NEURODEGENERATIVE DISEASES

Abstract

The present invention relates to a compound of the following formula (I): or a pharmaceutically acceptable salt or hydrate thereof, for use in the prevention or treatment of a neurodegenerative disease selected from the group consisting of a-synucleinopathy, in particular Lewy body disease, Huntington's disease, frontotemporal neurocognitive disorder, and amyotrophic lateral sclerosis in an individual.

##STR00001##

Claims

1.-16. (canceled)

17. A method for preventing or treating a neurodegenerative disease in an individual comprising administering to the individual an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or hydrate thereof, wherein the neurodegenerative disease is selected from the group consisting of a-synucleinopathy, in particular Lewy body disease, Huntington's disease, frontotemporal neurocognitive disorder, and amyotrophic lateral sclerosis, and wherein the compound of formula (I) has the following structure: ##STR00018## wherein: R.sub.1 represents —H or a ribosyl group of the following formula: ##STR00019## wherein: R.sub.6 represents —H; —O—R.sub.9 or —O—CO—R.sub.9 wherein R.sub.9 is H, an alkyl group having from 1 to 6 carbon atoms or an aryl group having from 3 to 12 carbon atoms; R.sub.7 represents —H; —O—R.sub.10 or —O—CO—R.sub.10 wherein R.sub.10 is H, an alkyl group having from 1 to 6 carbon atoms or an aryl group having from 3 to 12 carbon atoms; a deoxyribonucleic acid group; or a ribonucleic acid group; R.sub.8 represents —H; —O—R.sub.11 or —O—CO—R wherein R.sub.11 is H, an alkyl group having from 1 to 20 carbon atoms or an aryl group having from 3 to 20 carbon atoms; a phosphate group; a diphosphate group; a triphosphate group; a deoxyribonucleic acid group; or a ribonucleic acid group; and R.sub.12 represents a saturated or unsaturated alkyl, cycloalkyl, heterocycloalkyl or ether group having from 1 to 20 carbon atoms, optionally substituted by at least one group selected from the group consisting of: an alkyl group having from 1 to 20 carbon atoms, an aryl or heteroaryl group having from 3 to 20 carbon atoms, a cycloalkyl or heterocycloalkyl group having from 3 to 20 carbon atoms, a hydroxyl group, a carbonyl or carboxyl group having from 1 to 20 carbon atoms, an epoxy group, an —O—R.sub.4 group wherein R.sub.4 is H, an alkyl group having from 1 to 6 carbon atoms, an aryl group having from 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group, and an —O—CO—R.sub.5 group wherein R.sub.5 is an alkyl group having from 1 to 6 carbon atoms, an aryl group having from 3 to 12 carbon atoms or a glycosyl group.

18. The method of claim 17, wherein the compound of formula (I) is of the following formula (II): ##STR00020## wherein: a represents a double bond or an epoxy group, and R.sub.1 represents —H or a ribosyl group of the following formula: ##STR00021## wherein: R.sub.6 represents —H; —O—R.sub.9 or —O—CO—R.sub.9 wherein R.sub.9 is H, an alkyl group having from 1 to 6 carbon atoms or an aryl group having from 3 to 12 carbon atoms; R.sub.7 represents —H; —O—R.sub.1o or —O—CO—R.sub.10 wherein R.sub.10 is H, an alkyl group having from 1 to 6 carbon atoms or an aryl group having from 3 to 12 carbon atoms; a deoxyribonucleic acid group; or a ribonucleic acid group; R.sub.8 represents —H; —O—R.sub.11 or —O—CO—R.sub.11 wherein R.sub.11 is H, an alkyl group having from 1 to 20 carbon atoms or an aryl group having from 3 to 20 carbon atoms; a phosphate group; a diphosphate group; a triphosphate group; a deoxyribonucleic acid group; or a ribonucleic acid group; and R.sub.2 and R.sub.3, which are identical or different, represent —O—R.sub.4 wherein R.sub.4 is H, an alkyl group having from 1 to 6 carbon atoms, an aryl group having from 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group; or —O—CO—R.sub.5 wherein R.sub.5 is an alkyl group having from 1 to 6 carbon atoms, an aryl group having from 3 to 12 carbon atoms or a glycosyl group.

19. The method of claim 18, wherein: R.sub.2 and R.sub.3, which are identical or different, represent —OH, a —O-mannosyl group, a —O-galactosyl group or a —O-glutamyl group; R.sub.6 represents —OH; and R.sub.7 and R.sub.8, which are identical or different, represent —OH or a ribonucleic acid group.

20. The method of claim 17, wherein the compound is selected form the group consisting of queuine, ent-queuine, queuosine, epoxyqueuine, epoxyqueuosine, mannosyl-queuine, galactosyl-queuine, glutamyl-queuine, galactosyl-queuosine, mannosyl-queuosine, glutamyl-queuosine, queuine-tRNA and epoxyqueuine-tRNA.

21. The method of claim 17, wherein the compound is selected from the group consisting of the compounds of the following formulae: ##STR00022## ##STR00023##

22. The method of claim 17, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered at a unit dose of from 5 to 1,500 mg/kg.

23. The method of claim 17, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered at a dosage regimen of from 0.01 to 40 mg/kg/d.

24. The method of claim 17, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is formulated for administration by the oral route, the intradermal route, the intravenous route, the intramuscular route or the subcutaneous route.

25. The method of claim 17, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered in combination with at least one additional compound useful for the prevention or treatment of a neurodegenerative disease selected from the group consisting of a-synucleinopathy, in particular Lewy body disease, Huntington's disease, frontotemporal neurocognitive disorder, and amyotrophic lateral sclerosis.

26. The method of claim 25, wherein the at least one additional compound is selected from the group consisting of tetrabenazine, risperidone, olanzapine, citalopram, fluoxetine, L-Dopa, donepezil, galantamine, memantine, rivastigmine, trazodone, riluzole, edaravone, bromocriptine, cabergoline, pramipexole, ropinirole, pirebidil, lisurdide, apomorphine, selegiline, and rasagiline.

27. The method of claim 25, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered as a combined preparation with the at least one additional compound.

28. The method of claim 25, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered in a separate manner with respect to the at least one additional compound.

29. The method of claim 17, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered in association with at least one pharmaceutically acceptable excipient or vehicle.

30. The method of claim 17, wherein the compound or pharmaceutically acceptable salt or hydrate thereof is administered in the form of a dietary supplement.

31. The method of claim 30, wherein the dietary supplement further comprises additional compounds selected from the group consisting of vitamins, minerals, fatty acids, amino acids and antioxidants.

Description

DESCRIPTION OF THE FIGURES

[0195] FIG. 1

[0196] FIG. 1 represents an immunofluorescence microscope photograph of WT mouse cortical neurons exposed to 10 nM of huPFFs, stained with the EP1536Y antibody which specifically detects the S129 phosphorylated form of a-synuclein, and either pre-treated with 100 nM queuine (right panel) or not treated (control, left panel).

[0197] FIG. 2

[0198] FIG. 2 represents the quantification of the EP1536Y signal and Lewy neurite length after normalisation with the huPFF condition (huPFFs, no compound) in WT cortical neurones (vertical axis) as a function of queuine concentration (horizontal axis). The data are expressed as mean of group and SEM. Data were analysed using two-way ANOVA followed by Dunnett's multiple comparison; ***p<0.001 cf. huPFFs+0 μM group

EXAMPLE

[0199] The study was designed to evaluate the effects of queuine in an in vitro cell-based experimental model of synucleinopathy. The study aimed to evaluate the impact of queuine on the experimental synucleinopathy, in particular of the Lewy body type, induced by exogenous human preformed a-synuclein fibrils (huPFFs) in cortical cultures of wild-type (WT) mice (Volpicelli-Daley et al. (2011) Neuron. 72:57-7).

[0200] Material and Methods

[0201] Queuine was evaluated for its capability to afford protection against the induction of endogenous a-synuclein phosphorylation at serine 129 (a surrogate marker of de novo a-synuclein aggregation) triggered by exposure to exogenous human recombinant (unphosphorylated) a-synuclein preformed fibrils (huPFFs).

[0202] Primary cultures of dissociated cortices from embryonic day E18 WT mouse embryos were used as the biological system. Timed pregnant female mice were received from Charles River Laboratories 2 days before initiation of the primary culture. Cortices were harvested from the E18 embryos of WT mice and dissociated enzymatically and mechanically to yield a homogenous cell suspension. 20000 cells were plated per well in poly-D-Lysine-coated 96-well plates in a neuronal medium containing 0.5% Penicillin/Streptomycin and 0.5 mM L-glutamine. The cultures were incubated at 37° C./5% CO2. At DIV 3 they were exposed to queuine applied at 3 concentrations (10 μM, 1 μM, 0.1 μM) or zero. Queuine treatment was then renewed every 3 days (by replacing ⅓ of the volume of medium with fresh medium supplemented with 1× concentration of QUEUINE). At DIV 7, half of the culture wells were additionally exposed to huPFFs (at 1 concentration, i.e. equivalent to 10 nM a-synuclein monomer, final concentration). At 30 DIV, the neuronal cultures were fixed with PFA, and the effects of the treatments were evaluated by performing two double immunostainings: [0203] (i) D37A6/Syn1 to detect total rodent a-synuclein/human plus rodent non aggregated a-synuclein; [0204] (ii) EP1536Y/Syn211 to detect rodent and human a-synuclein phosphorylated at S129/total human a-synuclein.

[0205] The induction of a-synuclein phosphorylation was quantified by measuring the accumulation of phospho-a-synuclein (length of the phospho-a-synuclein-positive neurite network, number of phospho-a-synuclein positive cell bodies).

[0206] The experimental conditions were performed in quadruplicate (for compound-treated conditions+/−huPFFs) and triplicate (for controls+/−huPFFs) wells. An array of nine individual microscopic fields were acquired in each single well, and 3 channels were recorded for each field (green fluorescence excited @488 nm, red fluorescence excited @594 nm, and phase contrast).

[0207] Results

[0208] Endogenous a-Synuclein Detection

[0209] Immunofluorescence performed using D37A6 and syn-1 antibodies on WT mouse cortical cultures revealed the distribution and the amounts of endogenous murine a-synuclein. D37A6 specifically recognises the rodent form of the protein, principally expressed by neurones and distributed at the presynaptic sites, while it is less prone to hybridise with the phosphorylated/aggregated forms of the protein. The syn-1 antibody does not have a species specificity, but it is conformation sensitive: in non-denaturing conditions, it binds neither to the fibrillar (amyloid) form of the endogenous protein nor to the exogenously added huPFFs in which the epitope is hidden by its engagement into the amyloid structure.

[0210] In the case of the WT mouse primary neuronal cultures, both antibodies stain endogenous a-synuclein and their signals largely overlap.

[0211] The induction of a-synuclein de novo aggregation/phosphorylation upon exposure to huPFFs is thus expected to cause a decrease of the signals obtained by both the antibodies due to the recruitment of the detectable a-synuclein forms (non-amyloid ones) into less- or nondetectable aggregated assemblies (amyloid).

[0212] huPFFs Detection and Induced a-Synuclein Phosphorylation

[0213] Immunofluorescence performed using Syn-211 specifically allows the detection of human a-synuclein, even when engaged into amyloid structures (the only “invisible” forms are the Cter truncated ones). Thus, in the present experimental model this antibody could only bind the exogenous huPFFs, provided that human a-synuclein was not cleaved at its C-terminal. After three weeks of exposure, huPFFs were largely processed in the neurones and the syn211 signal tended to disappear. Human synuclein could, however, still be detected, associated with neurones, but also particularly with a non-neuronal subpopulation of cells (astrocytes) that accumulate huPFFs without processing them efficiently.

[0214] The EP1536Y antibody specifically detects the S129 phosphorylated form of a-synuclein (both human and rodent). In WT primary cultures of cortical neurones, a-synuclein phosphorylation is (i) specifically induced by PFFs exposure, (ii) associated with a-synuclein aggregation and (iii) is restricted to the endogenous protein (the huPFFs do not get phosphorylated at S129). EP1536Y staining shows the appearance of Lewy neurites and a-synuclein aggregates in the neuronal cell bodies, all of them caused by huPFFs exposure. No signal is observed in the controls.

[0215] Effects of Queuine on huPFFs Induced a-Synuclein Phosphorylation

[0216] Exposure of the neurones to 10 nM huPFFs for 21 or 23 days induced the phosphorylation of endogenous a-synuclein.

[0217] The EP1536Y immunofluorescence signal (in green), representative of the phosphorylation of a-synuclein, is decreased by queuine pretreatment at 100 nM compared to control (FIG. 1), thereby showing that queuine inhibits the phosphorylation of a-synuclein induced by huPFFs.

[0218] To further evaluate the effects of queuine, the EP1536Y signal was segmented to quantify the phospho-a-synuclein positive neurites. The results were normalised with regards to the huPFFs-treated condition to appreciate the effects of the compound on the PFFs-induced phosphorylation (FIG. 2).

[0219] In the concentration range tested, queuine had no effect on the untreated neurones, i.e. it did not induce any synuclein phosphorylation at S129. In huPFFs-treated neurones, and at all doses tested, queuine antagonised the effects of PFFs, and induced a significant decrease of a-synuclein phosphorylation at S129 (approx. −50%) induced by huPFFs.