COMPOUNDS FOR TREATING ALZHEIMER'S DISEASE

Abstract

The present invention relates to a compound of the following formula (I): (I) or a pharmaceutically acceptable salt or hydrate thereof, for use in the prevention or treatment of Alzheimer's disease in an individual.

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Claims

1-16. (canceled)

17. A method for treating or preventing Alzheimer's disease in an individual comprising administering to the individual queuine or a pharmaceutically acceptable salt or hydrate thereof.

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

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

20. The method of claim 17, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered orally, intradermally, intravenously, intramuscularly, or subcutaneously.

21. The method of claim 17, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in combination with at least one additional compound useful for treating or preventing Alzheimer's disease.

22. The method of claim 21, wherein the additional compound is selected from the group consisting of donepezil, galantamine, memantine, and rivastigmine.

23. A composition comprising (i) queuine or a pharmaceutically acceptable salt or hydrate thereof and (ii) an additional compound useful for treating or preventing Alzheimer's disease.

24. The composition of claim 23, further comprising a pharmaceutically acceptable excipient or vehicle.

25. The composition of claim 23, wherein the additional compound is selected from the group consisting of donepezil, galantamine, memantine, and rivastigmine.

26. The composition of claim 25, further comprising a pharmaceutically acceptable excipient or vehicle.

27. A method for causing a neuroprotective effect in an individual having Alzheimer's disease comprising administering to the individual queuine or a pharmaceutically acceptable salt or hydrate thereof.

28. The method of claim 27, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in a dosage regimen of from 0.01 mg/kg/d to 40 mg/kg/d.

29. The method of claim 28, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in a dosage regimen of from 0.01 mg/kg/d to 10 mg/kg/d.

30. The method of claim 29, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in a dosage regimen of from 0.01 mg/kg/d to l mg/kg/d.

31. The method of claim 30, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in a dosage regimen of from 0.01 mg/kg/d to 0.1 mg/kg/d.

32. The method of claim 27, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered orally, intradermally, intravenously, intramuscularly, or subcutaneously.

33. The method of claim 27, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in combination with a compound selected from the group consisting of donepezil, galantamine, memantine, and rivastigmine.

34. The method of claim 27, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is administered in the form of a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient or vehicle and queuine.

35. The method of claim 34, wherein the pharmaceutical composition further comprises a compound selected from the group consisting of donepezil, galantamine, memantine, and rivastigmine.

36. The method of claim 27, wherein the queuine or pharmaceutically acceptable salt or hydrate thereof is formulated as a dietary supplement.

37. The method of claim 36, wherein the dietary supplement comprises additional compounds selected from the group consisting of vitamins, minerals, fatty acids, amino acids, and antioxidants.

Description

DESCRIPTION OF THE FIGURES

[0136] FIG. 1

[0137] FIG. 1 shows the effect of queuine at 5 different concentrations (30 nM, 100 nM, 300 nM, 1 M, and 3 M) on cortical survival, expressed in percentage of vehicle group, after intoxication with A.sub.1-42. Each value represents the meansem (100%=no ABI-42). (*) represents p<0.05. BDNF (50 ng/ml) is the referenced compound.

[0138] FIG. 2

[0139] FIG. 2 shows the effect of queuine at 5 different concentrations (30 nM, 100 nM, 300 nM, 1 M, and 3 M) on the neurite network, expressed in percentage of vehicle group, after intoxication with A.sub.1-42. Each value represents the meansem (100%=no A$1-42). (*) represents p<0.05. BDNF (50 ng/ml) is the referenced compound.

[0140] FIG. 3

[0141] FIG. 3 show the effect of queuine at 5 different concentrations (30 nM, 100 nM, 300 nM, 1 M, and 3 M) on the ratio Tau/neurite, expressed in percentage of vehicle group, after intoxication with A.sub.1-42. Each value represents the meansem (100%=no A$1-42). (*) represents p<0.05. BDNF (50 ng/ml) is the referenced compound.

EXAMPLE 1: EVALUATION OF THE EFFECT OF COMPOUNDS ACCORDING TO THE INVENTION IN AN IN VITRO MODEL OF ALZHEIMER'S DISEASE

[0142] The purpose of this example is to evaluate the effects of compounds according to the invention in the in vitro model of Alzheimer's disease deriving from intoxication of cortical neurons by the amyloid .sub.1-42 peptide, in accordance with Callizot et al. (2013) J. Neurosci. Res. 91: 706-16; Chumakov et al. (2015) Nature Scientific Reports 5: 7608; Combs et al. (2000) J. Neurosci. 20:558-67; Cummings et al. (1998) Neurology. 51(Suppl 1): S2-17, discussion S65-7; Harrison (1990) J. Physiol. 422: 433-446; Kawahara et al. (2000) Brain Res. Bull. 53: 389-97; Pike et al. (1991) Brain Res. 563: 311-4; Sakono et al. (2010) FEBS J. 277: 1348-58; Singer et al. (1999) J. Neurosci. 19: 2455-2463; Sisodia et al. (1990) Science. 248: 492-5.

[0143] A. Materials and Methods

[0144] 1. Cortical Neuron Culture Rat cortical neurons are cultured as described by Singer et al. (1999) J. Neurosci. 19: 2455-2463. Briefly pregnant female rats of 15 days gestation are killed by cervical dislocation and the foetuses are removed from the uterus. The cortex is removed and placed in ice-cold medium of Leibovitz containing 2% of Penicillin and Streptomycin (PS) and 1% of bovine serum albumin. The cortex is dissociated by trypsinisation (0.05%) for 20 min at 37 C. The reaction is stopped by the addition of Dulbecco's modified Eagle's medium (DMEM) containing DNAase I grade II (0.1 mg/ml) and 10% of foetal calf serum (FCS). Cells are then mechanically dissociated by 3 passages through a 10 ml pipette. Cells are then centrifuged at 515g for 10 min at 4 C. The supernatant is discarded and the cells of pellet are re-suspended in a defined culture medium consisting of Neurobasal supplemented with 2% of B27 supplement, 2 mM of L-glutamine, 2% of PS solution and 10 ng/ml of BDNF. Viable cells are counted in a Neubauer cytometer using the trypan blue exclusion test. The cells are seeded at a density of 30 000 cells/well in 96 well-plates pre-coated with poly-D-lysine and are cultured at 37 C. in a humidified air (95%)/CO2 (5%) atmosphere.

[0145] 2. Effect of Queuine on Neuronal Cell Death after -Amyloid Injury in Cortical Neurons

[0146] 2.1. Incubation of Queuine 6 Days Before Intoxication

[0147] After 5 days of culture, the cells are incubated with queuine (7 concentrations). After 6 days of incubation with queuine, cells are intoxicated with 10 M of amyloid 1-42 oligomers in a medium as defined by Callizot et al. (2013) J. Neurosci. Res. 91: 706-16 in the presence of queuine. BDNF (50 ng/ml) is used as positive control and reference compound.

[0148] The following conditions are tested: [0149] Vehicle solution [0150] Amyloid .sub.1-42 at 10 M, 24h [0151] Amyloid .sub.1-42 at 10 M, 24h+BDNF (50 ng/mL) [0152] Amyloid-.sub.1-42 at 10 M, 24h+queuine (0.0001 M; 0.001 M; 0.01 M; 0.03 M; 0.100 M; 0.300 M; 1 M) added 6 days before intoxication.

[0153] Six wells per condition and 3 cultures are performed.

[0154] 2.2. Incubation of Queuine 24H Before the Intoxication

[0155] Briefly, on day 10, queuine (7 concentrations) is added. 24H after, cells are intoxicated with Amyloid .sub.1-42 at 10 M for 24h.

[0156] The following conditions are tested: [0157] Control medium [0158] Amyloid .sub.1-42 at 10 M, 24h [0159] Amyloid .sub.1-42 at 10 M, 24h+BDNF (50 ng/mL) [0160] Amyloid .sub.1-42 at 10 M, 24h+queuine (0.001 M; 0.01 M; 0.03 M; 0.100 M; 0.300 M; 1 M; 3 M) added 24H before intoxication.

[0161] Six wells per condition and 3 cultures are performed.

[0162] 3. End Point Evaluation: Measure of Total Number of Rat Cortical Neurons

[0163] 24 hours after intoxication, cells are fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min. The cells are then permeabilized and non-specific sites are blocked with a solution of phosphate buffered saline (PBS) containing 0.1% of saponin and 1% fetal calf serum (FCS) for 15 min at room temperature. Then, cells are incubated with monoclonal antibody anti microtubule-associated-protein 2 (MAP-2).

[0164] This antibody stains specifically cell bodies and neurites of neurons. This antibody is revealed with Alexa Fluor 488 goat anti-mouse IgG. Nuclei of neurons are labeled by a fluorescent marker (Hoechst solution).

[0165] The neuron survival is evaluated (number of MAP-2 positive neuronal cell bodies are counted).

[0166] For each well of culture, 20 pictures per well are taken using InCell Analyzer 2000 (GE Healthcare) with 20 magnification. All the images are taken in the same conditions. All values are expressed as means.e. mean. Statistical analyses are done on different conditions (ANOVA followed by Dunnett's test).

[0167] An increased survival of neurons incubated with queuine as compared to conditions without queuine indicates that queuine has anti-Alzheimer's disease properties.

EXAMPLE 2: IN VIVO EVALUATION OF THE EFFECT OF COMPOUNDS ACCORDING TO THE INVENTION IN A MURINE MODEL OF ALZHEIMER'S DISEASE BY ADMINISTRATION OF THE AMYLOID B25-35 PEPTIDE

[0168] The purpose of this example is the in vivo evaluation of compounds according to the invention in a murine model of Alzheimer's disease by administration of the amyloid $25-35 peptide in accordance with Maurice et al. (1996) Brain Res. 706:181-193; Maurice et al. (1998) Neuroscience 83:413-428; Meunier et al. (2006) J. Pharmacol. Exp. Ther. 317:1307-1319; Meunier et al. (2013) Genome Research 23:34-45; Villard et al. (2009) Neurophsychopharmacologie 34:1552-1566; Villard et al. (2011) J. Psychopharmacol. 25:1101-1117.

[0169] A. Materials and Methods

[0170] 1. Animals

[0171] Male Swiss mice, 5 weeks old and weighing 30-35 g (JANVIER, Saint Berthevin, France), are kept for housing. Animals are housed in groups with access to food and water ad libitum, except during behavioural experiments. They are kept in a temperature and humidity-controlled animal facility on a 12 h/12 h light/dark cycle (lights off at 07:00 pm). Mice are numbered by marking their tail using permanent markers.

[0172] 2. Protocol

[0173] Seventy-two male Swiss mice (30-35 g) are used. Six animal groups are constituted and submitted to different treatments:

TABLE-US-00001 TABLE 1 treatment groups Number of mice 1. Sc.A + vehicle solution, IP 12 2. A.sub.25-35 + vehicle, IP 12 3. A.sub.25-35 + reference compound 12 (Donepezil, 1 mg/kg), IP 4. A.sub.25-35 + queuine, IP (1 mg/Kg) 12 5. A.sub.25-35 + queuine, IP (5 mg/Kg) 12 6. A.sub.25-35 + queuine, IP (30 mg/Kg) 12 Total mice number 72

[0174] Day 1: a scrambled version of the amyloid $25-35 peptide (Sc.A, negative control) or the amyloid .sub.25-35 peptide (A25-35) is injected intracerebroventricularly (ICV) (the A.sub.25-35 peptide causes cellular intoxication). Between the 1st and 10th day, treatments are administered once a day intraperitoneally (IP).

[0175] At days 8 to 10, two different behavioral tests are used: [0176] Spontaneous alternation procedure in the Y-maze (assessing spatial working memory) at day 8 (7 days after the peptide injection); [0177] Passive avoidance response (assessing contextual long-term memory) with training at day 9 and retention session at day 10.

[0178] On day 10, after the behavioral test, animals are sacrificed by decapitation. Blood plasma samples are collected on each animal. The hippocampus and frontal cortex are dissected out and frozen in liquid nitrogen.

[0179] On n=6 animals per group, one hemi-hippocampus is used to determine the lipid peroxidation levels using a colorimetric method.

[0180] On n=6 animals per group, one hemi-hippocampus is used to determine the level of four biochemical markers using ELISA assays.

[0181] The other brain structures are stored at 80 C. and are available for supplementary biochemical assays.

[0182] 3. Products

[0183] 3.1. Compounds

[0184] Donepezil is solubilized in NaCl 0.9% [0185] Denomination: donepezil [0186] IUPHAR name: 2-[(1-benzyl-4-piperidyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one, hydrochloride, hydrate (1:1:1) [0187] CAS: 120014-06-4 [0188] Supplier: Sigma-Aldrich (France) [0189] Reference: D6821

[0190] A.sub.25-35: [0191] Denomination: amyloid-B protein (25-35), human, mouse, rat [0192] CAS: 131602-53-4 [0193] Supplier: Polypeptides (France) [0194] Reference: SC489

[0195] Sc.A: [0196] Denomination: scrambled amyloid-B protein (25-35), human, mouse, rat [0197] CAS: NA [0198] Supplier: Polypeptides (France) [0199] Reference: SC942

[0200] Queuine is obtained from Synthenova SAS, 15 rue J. Baptiste Lamarck, 14 200 Herouville Saint Clair.

[0201] 3.2. Amyloid Peptides Administration

[0202] Each mouse is anesthetized with isoflurane 2.5% and injected ICV with A.sub.25-3.sub.5 peptide (9 nmol/mouse) or Sc.A peptide (9 nmol/mouse), in a final volume of 3 L/mouse. These injections help to establish a mouse model of Alzheimer's disease according to Maurice et al. (1996) Brain Res. 706:181-193; Maurice et al. (1998) Neuroscience 83:413-428; Meunier et al. (2006) J. Pharmacol. Exp. Ther. 317:1307-1319; Meunier et al. (2013) Genome Research 23:34-45; Villard et al. (2009) Neurophsychopharmacologie 34:1552-1566; Villard et al. (2011) J. Psychopharmacol. 25:1101-1117.

[0203] 4. Conduct of the Tests

[0204] 4.1. Behavioral and Biochemical Analysis

[0205] 4.1.1. Spontaneous Alternation Performance

[0206] On day 8, all animals are tested for spontaneous alternation performance in the Y-maze, an index of spatial working memory. The Y-maze is made of grey polyvinylchloride. Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converging at an equal angle. Each mouse is placed at the end of one arm and allowed to move freely through the maze during an 8 min session. The series of arm entries, including possible returns into the same arm, is checked visually. An alternation is defined as entries into all three arms on consecutive occasions. The number of maximum alternations is therefore the total number of arm entries minus two and the percentage of alternation is calculated as (actual alternations/maximum alternations)100. Parameters included the percentage of alternation (memory index) and total number of arm entries (exploration index) (Maurice et al. (1996) Brain Res. 706:181-193; Maurice et al. (1998) Neuroscience 83:413-428; Meunier et al. (2006) J. Pharmacol. Exp. Ther. 317:1307-1319; Meunier et al. (2013) Genome Research 23:34-45; Villard et al. (2009) Neurophsychopharmacologie 34:1552-1566; Villard et al. (2011) J. Psychopharmacol. 25:1101-1117). Animals showing an extreme behavior (Alternation percentage <20% or >90% or number of arm entries <10) are discarded from the calculation.

[0207] 4.1.2. Passive Avoidance Test

[0208] The apparatus is a two-compartments (152015 cm high) box with one illuminated with white polyvinylchloride walls and the other darkened with black polyvinylchloride walls and a grid floor. A guillotine door separates each compartment. A 60 W lamp positioned 40 cm above the apparatus lights up the white compartment during the experiment. Scrambled footshocks (0.3 mA for 3 s) are delivered to the grid floor using a shock generator scrambler (Lafayette Instruments, Lafayette, USA). The guillotine door is initially closed during the training session. During training session, each mouse is placed into the white compartment. After 5 s, the door is raised. When the mouse entered the dark compartment and placed all its paws on the grid floor, the door is closed and the footshock delivered for 3 s. The step-through latency, that is, the latency spent to enter the dark compartment, and the number of vocalizations are recorded. The retention test is carried out 24 h after training. Each mouse is placed again into the white compartment. After 5 s, the door is raised. The step-through and escape latencies (corresponding to the re-exit from the dark compartment) are recorded up to 300 s (Meunier et al. (2006) J. Pharmacol. Exp. Ther. 317:1307-1319; Villard et al. (2009) Neurophsychopharmacologie 34:1552-1566; Villard et al. (2011) J. Psychopharmacol. 25:1101-1117).

[0209] Animals that showed latencies during the training and retention session both lower than 10 s are considered as failing to respond to the procedure and are discarded from the calculations.

[0210] 4.2. Lipid Peroxidation Measurement

[0211] All mice from each group are sacrificed by decapitation and both hippocampi are rapidly removed, weighed and kept in liquid nitrogen until assayed. After thawing, one hippocampus per mice is homogenized in cold methanol (1/10 w/v), centrifuged at 1,000 g during 5 min and the supernatant placed in Eppendorf tube. The reaction volume of each homogenate is added to FeSO4 1 mM, H2SO4 0.25 M, xylenol orange 1 mM and incubated for 30 min at room temperature. After reading the absorbance at 580 nm (A5801), 10 l of cumene hydroperoxide (CHP) 1 mM are added to the sample and incubated for 30 min at room temperature, to determine the maximal oxidation level. The absorbance is measured at 580 nm (A5802). The level of lipid peroxidation is determined as CHP equivalents according to: CHPE=A.sub.5801/A.sub.5802[CHP (nmol)] and expressed as CHP equivalents per mg of tissue and as percentage of control group data (V-treated Sc.A-administered mice).

[0212] 4.3. ELISA Assays

[0213] Contents in Bax, Bcl2, GFAP and Caspase-3 are analyzed by ELISA assays.

[0214] The kits are:

[0215] Mitochondrial Dysfunction/Apoptosis

[0216] Caspase-3: Supplier: USCNK Reference: SEA626Mu

[0217] Bax: Supplier: USCNK Reference: SEB343Mu

[0218] Bcl2: Supplier: USCNK Reference: SEA778Mu

[0219] Inflammatory Processes

[0220] GFAP: Supplier: USCNK Reference: SEA425Mu

[0221] For all assays, the cortices are homogenized after thawing in a Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.5) and sonicated for 20 s. After centrifugation (16,100 g for 15 min, 4 C.), supernatants are used for ELISA assays according to the manufacturer instructions. For each assay, absorbance is read at 450 nm and sample concentration is calculated using the standard curve. Results are expressed in pg or ng of marker per mg of tissue and in percent of Sc.A+Vehicle solution. 6 samples per group (n=36/ELISA kit) are assayed in duplicates.

[0222] An improvement of the tested behavioral and biochemical characteristics of mice administered with queuine as compared to mice which did not receive queuine indicates that queuine has anti-Alzheimer's disease properties.

EXAMPLE 3: EVALUATION OF THE EFFECT OF COMPOUNDS ACCORDING TO THE INVENTION IN AN IN VITRO MODEL OF ALZHEIMER'S DISEASE

[0223] The purpose of this example is to evaluate the effects of compounds according to the invention in the in vitro model of Alzheimer's disease deriving from intoxication of cortical neurons by the amyloid .sub.1-42 peptide, in accordance with Callizot et al. (2013) J. Neurosci. Res. 91: 706-16; Chumakov et al. (2015) Nature Scientific Reports 5: 7608; Combs et al. (2000) J. Neurosci. 20:558-67; Cummings et al. (1998) Neurology. 51(Suppl 1): S2-17, discussion S65-7; Harrison (1990) J. Physiol. 422: 433-446; Kawahara et al. (2000) Brain Res. Bull. 53: 389-97; Pike et al. (1991) Brain Res. 563: 311-4; Sakono et al. (2010) FEBS J. 277: 1348-58; Singer et al. (1999) J. Neurosci. 19: 2455-2463; Sisodia et al. (1990) Science. 248: 492-5, Jan et al., (2010) Nat. Protoc., 5: 1186-1209.

[0224] A. Material and Method

[0225] 1. Cortical Neurons

[0226] Rat cortical neurons are cultured as described by Callizot et al., (2013) with modifications. Briefly pregnant female rat (Wistar) of 15 days of gestation are killed. Foetuses are collected and immediately placed in ice-cold L15 Leibovitz medium with a 2% penicillin (10,000 U/mL) and streptomycin (10 mg/ml) solution (PS) and 1% bovine serum albumin (BSA). Cortex are treated for 20 min at 37 C. with a trypsin- EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociation is stopped by addition of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L of glucose, containing DNAse I grade II (final concentration 0.5 mg/ml) and 10% fetal calf serum (FCS). Cells are mechanically dissociated by three forced passages through the tip of a 10-ml pipette. Cells are then centrifuged at 515g for 10 min at 4 C. The supernatant is discarded, and the pellet is resuspended in a defined culture medium consisting of Neurobasal medium with a 2% solution of B27 supplement, 2 mmol/litter of L-glutamine, 2% of PS solution, 10 ng/mL of brain-derived neurotrophic factor (BDNF), 2% of heat-inactivated horse serum, 2% of heat-inactivated FCS, 1 g/L of glucose, 1 mM of sodium pyruvate, and 100 M of non-essential amino acids. Viable cells are counted in a Neubauer cytometer, using the trypan blue exclusion test. The cells are seeded at a density of 45,000 per well in 96-well plates (for immunostaining) precoated with poly-L-lysine and are cultured at 37 C. in an air (95%)-CO2 (5%) incubator. For 96 wells plates, only 60 wells are used. The wells of first and last lines and columns are not used (to avoid the edge effect) and are filled with sterile water. The medium is changed every 2 days.

[0227] 2. Test Compound and Human A.sub.1-42Exposure

[0228] On day 10 of culture, queuine is dissolved in the culture medium and pre-incubated with primary cortical neurons for 24H, before A.sub.1-42 exposure. Then on day 11 of culture, the cortical neurons are intoxicated with AB solutions. The A.sub.1-42 preparation is done following the procedure described by Callizot et al., (2013). Briefly, A.sub.1-42 peptide are dissolved in the defined culture medium mentioned above, at an initial concentration of 40 mol/L. This solution is gently agitated for 3 days at 37 C. in the dark and immediately used after being properly diluted in culture medium to the concentration used (5 M, 0.5M oligomers).

[0229] A.sub.1-42 preparation is added to a final concentration of 5M (0.5 M oligomers, ABO) diluted in control medium in presence of queuine, for 72 hours.

[0230] 3. Organization of Culture Plates

[0231] Each compound is tested on 1 culture in a 96 well plate (6 wells per conditions). For 96 wells plates, only 60 wells are used. The wells of first and last lines and columns are not used (to avoid the edge effect) and are filled with sterile water. Queuine is added 24h before A.sub.1-42 application. The following conditions are assessed:

TABLE-US-00002 Plate 1 (MAP-2/Tau phospho/OX-41) Control (vehicle) + A.sub.1-42 (5 M 72 H)/vehicle + A.sub.1-42 (5 M 72 H)/queuine (30 nM) + A.sub.1-42 (5 M 72 H)/queuine (100 nM) + A.sub.1-42 (5 M 72 H)/queuine (300 nM) + A.sub.1-42 (5 M 72 H)/queuine (1 M) + A.sub.1-42 (5 M 72 H)/queuine (3 M) + A.sub.1-42 (5 M 72 H)/BDNF (50 ng/mL)

[0232] 4. Evaluation

[0233] 4.1. Immunostaining

[0234] 72 hours after A.sub.1-42 application, the cell culture supernatant are collected cytokine quantification (e.g. TNF) and the cortical neurons are fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 min at 20 C. After permeabilization with 0.1% of saponin, cells are incubated for 2 hours with: [0235] a) chicken polyclonal antibody antimicrotubule-associated-protein 2 (MAP-2) at dilution of 1/1000 in PBS containing 1% fetal calf serum and 0.1% of saponin (this antibody stains specifically cell bodies and neurites, allowing study of neuronal cell death and neurite network). [0236] b) mouse monoclonal anti-tau phosphor (Thr212, Ser214) at the dilution of 1/100 in PBS containing 1% foetal calf serum and 0.1% of saponin. [0237] c) rabbit polyclonal antibody anti-SIRP alpha/CD172a at dilution of 1/400 in PBS containing 1% fetal calf serum and 0.1% of saponin (this antibody stains specifically the microglia and is allow to evaluate the activation of the microglial cells).

[0238] These antibodies are revealed with secondary antibodies goat anti-rabbit IgG, goat anti-mouse IgG and goat anti-chicken IgG coupled with an Alexa Fluor at the dilution 1/400 in PBS containing 1% FCS, 0.1% saponin, for 1 hour at room temperature.

[0239] For each condition, 30 pictures/well (representative of the whole well area) are automatically taken using ImageXpress (Molecular Devices) at 20 magnification. All images are automatically acquired under the same conditions. The following analysis is performed with Custom Module Editor (Molecular Devices): [0240] total neuron survival (number of MAP-2 positive neuron) [0241] total neurite network (length of MAP-2 positive neurite) [0242] hyperphosphorylation of Tau in neuron cytoplasm (AT100, overlapping Tau/MAP-2, m.sup.2 of overlapping) [0243] total microglia activation (area of microglial cells, m.sup.2 of OX-41 staining).

[0244] 4.2 TNF- Evaluation

[0245] In order to assess the activation of microglia in the cell culture, the levels of TNF- are quantified in cell culture supernatant after 72h (end of the culture) by ELISA.

[0246] 5. Statistical Analysis

[0247] All values are expressed as mean+/SEM. Statistical analysis are performed by one-way ANOVA, followed by Dunnett's or a PLSD Fisher test, p<0.05 is considered significant.

[0248] 6. Results

[0249] FIG. 1 shows that the number of cortical neurons decreases significantly in the presence of the peptide A$1-42 (5 M), compared to the control conditions. BDNF (50 ng/ml) restores the level neuronal staining for MAP-2.

[0250] Queuine has a neuroprotective effect on cortical neurons intoxicated with A.sub.1-42 peptide. Indeed, queuine significantly restores the survival of neurons at 100 nM (*, p <0.05), 300 nM (*, p<0.05) and 1 M (*, p<0.05).

[0251] FIG. 2 shows that the neurite network decreases in the presence of the peptide A.sub.1-42 (5 M) compared to the control conditions. BDNF (50 ng/ml) restores the neurite network. Queuine has a neuroprotective effect on neurons intoxicated with A.sub.1-42 peptide. Indeed, queuine significantly restores the neurite network at 300 nM (*, p <0.05), 1 M (*, p<0.05) and 3 M (*, p<0.05).

[0252] FIG. 3 shows that the peptide A.sub.1-42 (5 M) induces a significant increase in Tau hyperphosphorylation. BDNF (50 ng/ml) decreases Tau hyperphosphorylation. Queuine at 100 nM (*, p<0.05), 300 nM (*, p<0.05), and 1 M (*, p<0.05) and 3 M (*, p<0.05) restores the level of hyperphosphorylation of Tau.

[0253] The results of the TNF- evaluation are shown in the in following Table 1:

TABLE-US-00003 TABLE 1 TNFa release in a culture of cortical neurons and microglia after A.sub.1-42 injury (5 M, 72 H) A.sub.1-42 A.sub.1-42 (5 M) + A.sub.1-42 (5 M) + A.sub.1-42 (5 M) + A.sub.1-42 (5 M) + Control (5 M) queuine (300 nM) queuine (1 M) queuine (3 M) BDNF (50 ng/ml) TNFa (% of 100.00 126.75 105.54 87.33 93.32 102.14 vehicle group)

[0254] Table 1 shows that the peptide A.sub.1-42 (5 M) induces a significant increase in TNF. BDNF (50 ng/ml) decreases the level of TNF. Queuine at 300 nM (*, p<0.05), 1 M (*, p<0.05), and 3 M (*, p<0.05) restores the level of TNF.