Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4709—Amyloid plaque core protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• the present invention relates to the medical field, and is of particular interest to pharmacological research units.
• the invention relates in fact to a method for screening compounds with anti-amyloid properties.
• the invention implements biochemical techniques for the ex vivo analysis of biological samples making it possible to rapidly identify compounds intended for the curative and / or preventive treatment of neurodegenerative diseases and of Alzheimer's disease in particular .
• the subject of the invention is a method for screening compounds having the ability to dissociate high affinity complexes between the ⁇ -amyloid peptide and the nicotinic acetylcholine receptor of human cortex tissues.
• Alzheimer's disease is a progressive neurodegenerative disease that affects a large proportion of the elderly population. This disease is clinically characterized by a loss of memory and a decline in cognitive function. Neuropathologically, Alzheimer's disease is characterized by the presence of two types of brain histopathological lesions: amyloid plaques and neurofibrillary degeneration (DNF). A third characteristic of Alzheimer's disease is cortical atrophy corresponding to pronounced neuronal loss.
• DNF neurofibrillary degeneration
• a ⁇ ⁇ -amyloid peptides
• amyloid plaques or senile plaques around the neurons would be at the origin of the etiology of Alzheimer's disease.
• amyloid deposits represents the early and invariable characteristic of all forms of Alzheimer's disease, including familial forms.
• Neurofibrillary degeneration is an intraneuronal accumulation of fibrils formed of helically paired filaments or PHFs (paired helical filaments).
• PHFs are constituted by the assembly of tau microtubular proteins. The biochemical characterization of these proteins reveals the presence of a major triplet of tau proteins abnormally phosphorylated (tau 60, 64 and 69) and aggregated.
• Normal tau protein is phosphorylated 2 to 3 times as opposed to 5 to 9 times in Alzheimer's disease and plays a role in the polymerization-depolymerization of microtubules of the neuronal cytoskeleton as well as in axonal transport.
• Cortical atrophy results in a loss of 8 to 10% of brain weight every 10 years in patients with Alzheimer's disease, while in healthy subjects this loss is only 2%. Cortical atrophy is accompanied by dilated cerebral ventricles and cortical furrows, reduced hippocampal volume, and neuronal loss, particularly affecting the cholinergic system.
• the amyloid plaques result from spherical deposits of amyloid substance.
• the amyloid substance consists of filaments of a polypeptide of 39 to 43 amino acids called A ⁇ ( ⁇ -amyloid).
• the ⁇ -amyloid peptide has a ⁇ -sheet conformation conferring on it its insoluble character and its toxicity.
• the ⁇ -amyloid peptide is a normal catabolic product of a large membrane glycoprotein called APP (amyloid precursor protein).
• APP amyloid precursor protein
• the amyloid plaques are surrounded by neuritic extensions and glial cells. The amyloid plaques impregnate the nervous parenchyma and diffuse into the cortical gray matter of all brain regions. The occipital cortex seems more frequently affected by these amyloid deposits.
• the neurotoxicity of ⁇ -amyloid peptide is a major problem of Alzheimer's disease.
• amyloid plaques located in the extracellular space are derived from cell lysis of neurons with a very large accumulation of amyloid deposits in the lysosomal compartment. This intraneuronal accumulation causes degeneration of the neuronal cell and cell death and release of these deposits in the extracellular space, gradually forming the amyloid plaques (Nagele et al., 2002). Amyloid plaques are surrounded by neuritic extensions and glial cells and contain fragments of nuclei, evidence that they come from dead neurons. The ⁇ 7-type nicotinic receptor plays a key role in the entry of A ⁇ peptide into neurons (D'Andréa and Nagele 2006). Wang et al.
• a ⁇ peptide binds specifically and with high affinity to ⁇ 7 nicotinic acetylcholine receptors ( ⁇ 7 nAChR) present at the extracellular surface of the neuron (Wang et al., 2000).
• the interaction of the A ⁇ peptide, in particular the A ⁇ 42 peptide, with the ⁇ 7 nAChR receptor appears to be an essential and prior stage for the intraneuronal accumulation of the A ⁇ 42 - ⁇ 7 nAChR complexes, said complexes on the surface of the neurons being subjected to endocytosis leading to their accumulation in the lysosomal compartment (Nagele et al., 2002).
• a first method consists of an injection of ⁇ -amyloid peptides in brains of mice, performed using a cannula ⁇ in the intra-cerebroventricular (icv) position.
• This method (Yamada et al 2005, Mazzola et al., 2003) makes it possible to obtain mice with a memory deficit after 7 days of exogenous supply of ⁇ -amyloid peptides.
• This mouse model is obtained quickly and can be used to test new products in the treatment of neurodegenerative diseases and Alzheimer's disease in particular.
• This method is based on a model that does not exactly represent the pathophysiology of Alzheimer's disease.
• this method of identifying compounds acting on the A ⁇ 42 - ⁇ 7 nAChR complex has no apparent validity since the tau pathology of cerebral aging does not develop in this mouse model.
• the present murine model does not respect a construction validity, given that, on the one hand, the ⁇ -amyloid peptides are of exogenous origin and not naturally produced by the animal and, on the other hand, the model is animal and not human.
• the injection of exogenous ⁇ -amyloid peptides into mouse brains requires working in vivo which rules out this method of routine use for the screening and identification of anti-Alzheimer compounds.
• transgenic mice as a model of Alzheimer's disease, carrying mutations present in familial forms of Alzheimer's disease, on APP and / or PS1 (presenilin-1) genes. These models therefore have an undeniable construction validity for family forms but very questionable for sporadic forms that represent more than 97% of cases.
• a first type of transgenic mouse has only one mutation on APP (Hsiao et al., 1996) or a double mutation on APP and PS1 (Holcomb et al., 1998).
• the descriptive validity of the transgenic models described above is not complete since they do not reliably reproduce the physiopathological characteristics associated with Alzheimer's disease. Indeed, we observe on the one hand an absence of degeneration neurofibrillary, on the other hand little or no neuronal loss as well as late onset of senile plaques in the cortex of single or double transgenic mice. Consequently, the use of single or double transgenic mouse models is not recommended in view of the physiological differences existing with Alzheimer's disease and the delay at which the lesions associated with this pathology appear.
• transgenic mouse model comprising three mutated genes (APP, PS1 and tau) (LaFerla et al., 2003) also has disadvantages.
• the construction validity of this model is questionable since a mutation in the tau gene, not present in humans with Alzheimer's disease, is added compared to previous models.
• the descriptive validity of this model is good since it mimics the physiological lesions of Alzheimer's disease that consist of amyloid plaques, neurofibrillary tangles and neuronal loss.
• this method requires a delay of 6 months to 12 months before obtaining mice with the typical lesions of Alzheimer's disease. Consequently, this transgenic mouse model can be validly used in a method for confirming the anti-amyloid properties of a test compound but can not reasonably be used as a first-line for the screening of compounds in view of the duration and the difficulty of implementing said model.
• a third method (Wang et al., 2000) is to test the ability of compounds to prevent the formation of exogenously delivered A ⁇ peptide complexes and ⁇ 7 present in rat tissues (hippocampal and rat cortex synaptosomes).
• This in vitro method is more rapidly implemented than the methods described above, however it has the disadvantage of not being representative of the complexes present in humans since they are extracts of rat brains and A ⁇ peptides are provided. exogenous way. Consequently, this model does not respect the conditions of validity of construction nor those of descriptive validity required for the use of the latter in a method of screening compounds capable of acting on the complex A ⁇ 42 - ⁇ 7 nAChR at the origin amyloid plaque formation.
• the object of the present invention is therefore to propose an alternative strategy to the methods for identifying compounds capable of acting on the ⁇ -amyloid- ⁇ 7 nAChR complexes, in order to remedy at least in part the drawbacks already known from the selection methods. said compounds.
• the invention therefore proposes an ex vivo screening method recreating the physiological conditions present in patients suffering from Alzheimer's disease. These optimal conditions are obtained by using human brains, in particular frontal cortex derived from patients suffering from Alzheimer's disease. This model fulfills by definition the criteria of validity of construction and descriptive validity since it is a direct use of the diseased human tissue.
• the ex vivo conditions of the screening method according to the invention make it possible to remove the constraints related to the production and manipulation of animal models.
• human biological materials makes it possible to overcome all the artifacts and errors related to the physiological differences existing between animal and human species.
• the use of human biological materials in the context of the screening method according to the invention is particularly important in view of the fact that Alzheimer's disease and neurodegenerative pathologies in general do not exist naturally in other species. than the human species.
• the invention thus relates to a method for screening compounds capable of dissociating or preventing ⁇ -amyloid peptide complexes with nicotinic acetylcholine receptors derived from human brains.
• the invention relates to a method for screening compounds capable of dissociating or preventing ⁇ -amyloid peptide complexes with ⁇ 7 nicotinic acetylcholine receptors derived from human brains.
• the screening method according to the invention therefore makes it possible to identify compounds with curative or preventive properties depending on whether these compounds are respectively capable of dissociating or preventing the A ⁇ 42 - ⁇ 7 nAChR complexes.
• anti-amyloid or “anti-beta amyloid” property is meant the ability for a compound to dissociate or oppose the formation of intracellular or extracellular deposits of ⁇ -amyloid peptides either by dissociation or by inhibition of the formation of the complexes formed by the A ⁇ peptides with the nicotinic acetylcholine receptors.
• alpha 7 nicotinic acetylcholine receptor denotes a pentameric cell surface receptor, expressed mainly in the cortex and the hippocampus, and having an important role in learning and memory. .
• ⁇ -amyloid refers to all of the ⁇ -amyloid peptides whose peptides A ⁇ 1-39 or A ⁇ 39 , A ⁇ 1-40 or A ⁇ 40 , A ⁇ ].
• the fragments of ⁇ -amyloid peptides supra have a biological activity and can be used in the screening method according to the present invention. Said fragments are, for example, fragments A ⁇ 1-28 and A ⁇ 25-35 .
• the ⁇ -amyloid peptides used in the context of the invention are in particular the peptides A ⁇ 3 ç > , A ⁇ 40 , A ⁇ 41 , A ⁇ 42 and / or A ⁇ 43 .
• a ⁇ 42 has the highest affinity for nicotinic ⁇ 7 acetylcholine receptors and has the most important role in the etiology of Alzheimer's disease.
• the present screening method is performed from human brain samples and preferably from human cortex and hippocampus. These samples are taken post-mortem from patients with Alzheimer's disease.
• the invention relates, preferably, to a screening method characterized in that the dissociation of ⁇ -amyloid peptide complexes and nicotinic ⁇ 7 receptors of acetylcholine is demonstrated by immunohistochemistry.
• immunohistochemistry relates to all techniques for the revelation of antigens by antibodies to detect or isolate defined molecules.
• the screening method comprises the following steps of incubation of ⁇ -amyloid peptide complexes with nicotinic acetylcholine receptors in presence or in the absence of a test compound, then determining the amount of undissociated complexes in the presence or absence of test compound and the evaluation of the difference in the amount of undissociated complexes, said difference indicating that the test compound modulates the dissociation of ⁇ -amyloid peptide complexes with nicotinic acetylcholine receptors.
• the screening method according to the invention also comprises a step of isolating the non-dissociated complexes of ⁇ -amyloid peptides with the nicotinic acetylcholine receptors using antibodies to the ⁇ -amyloid peptide.
• the anti- ⁇ -amyloid peptide antibodies used in the screening method are directed against the ⁇ -amyloid peptides A ⁇ 39 , A ⁇ 40 , A ⁇ 41 , A ⁇ 42 and / or A ⁇ 43 .
• These antibodies may be monoclonal antibodies of mouse or goat.
• the present screening method is characterized by the revelation, in particular by Western-blot method, of undissociated complexes using anti-nicotinic acetylcholine receptor antibodies, in particular anti-antibodies. nicotinic ⁇ 7 receptors of acetylcholine.
• the screening method according to the invention has demonstrated compound S 24795, chloride or iodide of 1- (4-bromophenyl) -2- (1-methyl-2-pyridiniumyl) -1-ethanone, as compound capable on the one hand, to inhibit the formation of ⁇ -amyloid- ⁇ 7 nAChR complexes and, on the other hand, to dissociate said ⁇ -amyloid- ⁇ 7 nAChR complexes accumulated in amyloid plaques around the neuron and in deposition within the neuron.
• the compound S 24795, identified by the screening method of the present invention is therefore a compound capable of dissociating the ⁇ -amyloid peptide complexes with the nicotinic acetylcholine receptors present in the brains of patients suffering from the disease. Alzheimer's as well as to inhibit the formation of said complexes.
• the invention also relates to each compound identified from the screening method according to the invention.
• the invention also relates to a pharmaceutical composition comprising the compound obtained from the screening method according to the invention as active principle in combination with one or more pharmaceutically acceptable excipients.
• active principle is meant any substance responsible for the pharmacodynamic or therapeutic properties of the pharmaceutical composition.
• excipients is understood to mean any substance into which the active principle of a medicament is incorporated in order to facilitate the preparation and administration thereof and to condition the consistency thereof, the form and than the volume.
• non-toxic, pharmaceutically acceptable excipients mention may be made, by way of indication and not limitation, of diluents, solvents, preservatives, wetting agents, emulsifiers, dispersing agents, binders, blowing agents, disintegrating agents, retardants, lubricants, absorbents, suspending agents, dyes or flavoring agents.
• compositions intended for the prevention and / or treatment of neurodegenerative diseases and Alzheimer's disease in particular are in a form suitable for oral, parenteral, nasal, percutaneous, rectal, perlingual, ocular or respiratory and in particular simple or coated tablets, sublingual tablets, sachets, packets, capsules, glossettes, lozenges, suppositories, creams, ointments, dermal gels, and oral or injectable ampoules.
• the present invention further relates to the use of compounds identified from the screening method according to the invention for obtaining pharmaceutical compositions for the prevention and / or treatment of neurodegenerative diseases.
• the compounds identified by the screening method according to the invention are used in the treatment of "neurodegenerative diseases” such as, for example, Alzheimer's disease, Pick's disease, Lewy body dementia, Steel-Ridchardson's syndrome. Down syndrome, Shy-Drager syndrome, amyotrophic lateral sclerosis, neurodegenerative ataxia, Huntigton's disease, Parkinson's disease, progressive primary aphasia, Machado-Joseph's disease, Parkinson's disease Gilles de La Tourette, paralytic parenchymia, Kennedy's disease, familial spasmodic paralysis, Werdriig-Hoffinann's disease, Kugelberg-Welander's disease, Tay-Sach's disease, Sandhoff's disease, Wohlfart's disease Kugelberg-Welander, spastic paraparesis, progressive multifocal leukoencephalitis and prion-related diseases including Creutzfeldt-Jakob or Gerstmann-Straus
• preventive corresponds to a preventive treatment intended to reduce the risk of developing Alzheimer's disease by inhibiting the binding of ⁇ -amyloid peptides on nicotinic cell receptors ⁇ 7 acetylcholine. This inhibition limits the formation of ⁇ -amyloid- ⁇ 7 nAChR complexes at the origin of amyloid plaques, a lesion present in Alzheimer's disease.
• preventive may be understood as secondary prevention that is intended to decrease prevalence by reducing the course and duration of the disease.
• treatment is meant a curative treatment prescribed for the purpose of treating patients with Alzheimer's disease by dissociating the ⁇ -amyloid- ⁇ 7 nAChR complexes present in the human brains and constituting the senile plaques.
• Alzheimer's disease is understood to mean a fatal neurodegenerative disease affecting memory and mental functioning, in particular with the alteration of language, the disruption of elaborate gestures and disorders of orientation in time and space. These cognitive disorders are related to two neuropathological lesions characteristic of senile plaques and neurofibrillary degeneration that allow definitive post-mortem diagnosis.
• FIG. 1 Western blot illustrating the inhibition by S 24795 of A ⁇ 42 - ⁇ 7 nAChR complexes originating from synaptosomes of frontal cortex of patients suffering from Alzheimer's disease or post-mortem control subjects.
• the A ⁇ 42 - ⁇ 7 nAChR complexes are incubated in medium alone (Krebs-Ringer) or in the presence of S 24795 (30 ⁇ M) for 10 minutes followed by incubation in the presence of A ⁇ peptide 42 for 30 minutes.
• FIG. 1 Western blot illustrating the inhibition by S 24795 of A ⁇ 42 - ⁇ 7 nAChR complexes originating from synaptosomes of frontal cortex of patients suffering from Alzheimer's disease or post-mortem control subjects.
• the A ⁇ 42 - ⁇ 7 nAChR complexes are incubated in medium alone (Krebs-Ringer) or in the presence of S 24795 (30 ⁇ M) for 10 minutes followed by incubation in the presence of A ⁇
• FIG. 3 Western blot illustrating the dissociation by S 24795 of A ⁇ 42 - ⁇ 7 nAChR complexes originating from synaptosomes of frontal cortex of patients suffering from Alzheimer's disease or from post-mortem control subjects.
• Complex Aß 42 - ⁇ 7 nAChR are incubated in the medium (Krebs-Ringer) or presence of S 24795 (1, 10, 30 or lOO ⁇ M) for 10 minutes and in the presence or in the absence of Aß 42
• Figure 5 Quantification of 45 Ca 2+ entry in synaptosomes of human frontal cortex from patients with Alzheimer's disease and post-mortem control subjects treated with S 24795. Control brain slices are incubated or not in the presence of A ⁇ 42 (1 ⁇ M) prior to treatment with S 24795 (10 ⁇ M). Calcium inputs are induced either by the ⁇ 7 agonist (PNU282987) or by NMDA added to glycine.
• Post mortem human frontal cortex from patients with Alzheimer's disease and healthy control subjects are from the Harvard Brain Tissue Resource Center and Analytical Biological Services.
• patients with Alzheimer's disease have been divided into two subgroups with or without associated vascular pathologies. Only the brains of patients without associated pathology were used in this study.
• the cortex collected in the study comes from people who died within 15 hours of sampling.
• the cortex removed are stored at -80 ° C until used in the screening method according to the invention.
• the cortex is cryoprotected for 2 weeks in 0.2M sodium phosphate buffer (NaH 2 PO 4 , 2H 2 O 2 NaH 2 PO 4 , 12H 2 O, pH 7.4) containing 20% (W / V) sucrose. . They are then frozen for 1 minute in isopentane maintained at a temperature of -30 ° C. in dry ice. Sections of 5 .mu.m thick, made in a cryostat thermostated at -30 ° C. (Super Frost Plus Fisher) are finally placed in a 0.02M PBS buffer and then stored at 4 ° C.
• a homogenization solution containing 25 mM HEPES at pH 7.5, 1 mM EDTA, 50 ⁇ g / ml leupeptin, 10 ⁇ g / ml aprotinin, 2 ⁇ g / ml of inhibitor soybean trypsin, 0.04mM PMSF, a mixture of phosphatase inhibitory proteins and 0.2% 2-mercaptomethanol.
• the homogenate is centrifuged at 1000 g and 4 ° C for 10 minutes. The supernatant from this first centrifugation is then centrifuged at 15000 g for 30 minutes in order to obtain a pellet of synaptosomes.
• the pellet of synaptosomes is washed twice by suspension in 10 ml of an ice-maintained Krebs-Ringer solution comprising 25 mM HEPES at pH 7.4, 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO 3 , 1.3 mM CaCl 2 1.2mM MgSO 4 , 1.2mM KH 2 PO 4 , 10mM glucose, 100 ⁇ M ascorbic acid, 50 ⁇ g / ml leupeptin, 10 ⁇ g / ml aprotinin, 2 ⁇ g / ml of soy trypsin inhibitor, 0.04mM PMSF and a mixture of phosphatase inhibiting proteins, aerated for 10 minutes with 95% O 2 /5% CO 2 and then centrifuged again at 15000g for 10 minutes at 4 ° C.
• the washed synaptosomes are then suspended in ImI of oxygenated Krebs-Ringer solution and the protein concentration of said synaptosome suspension
• the synaptosomes of human cortex are incubated in an oxygenated Krebs-Ringer solution in the presence of compound S 24795 at 37 ° C. for 30 minutes in a total incubation volume of 500 ⁇ l.
• Compound S 24795 is present in the reaction medium at concentrations of 1 ⁇ M, 10 ⁇ M, 30 ⁇ M or 100 ⁇ M.
• the synaptosomes are also incubated in the presence of 10 nM A ⁇ 42 or in the presence of a vehicle.
• the reaction is stopped by diluting in 1.5 ml of a 1M EDTA solution maintained in ice-Ca 2+ calcium ion - without Krebs-Ringer solution and then centrifuging for 10 minutes at 15000g and 4 ° C.
• the resulting synaptosome pellet is solubilized in 250 ⁇ l of immunoprecipitation buffer (25mM HEPES at pH 7.5, 20OmM NaCl, 1mM EDTA, 50 ⁇ g / ml leupeptin, 10 ⁇ g / ml aprotinin, 2 ⁇ g / ml soy trypsin inhibitor, 0.04mM PMSF and a mixture of inhibitors protein phosphatase) containing 0.5% digitonin, 0.2% chelated sodium and 0.5% NP-40.
• immunoprecipitation buffer 25mM HEPES at pH 7.5, 20OmM NaCl, 1mM EDTA, 50 ⁇ g / ml leupeptin, 10 ⁇ g / ml aprotinin, 2 ⁇ g / ml soy trypsin inhibitor, 0.04mM PMSF and a mixture of inhibitors protein phosphatase
• the A ⁇ 42 - ⁇ 7 nAChR complexes are isolated by immunoprecipitation using anti-A ⁇ antibodies. 42 incubated in their presence for 16 hours at 4 ° C and concentrated by incubation for 2 hours in the presence of 25 .mu.l of conjugated agarose beads A / G (Cai et al. 1999, Wang et al., 2000, Jin et al. 2001).
• isolated A ⁇ 42 - ⁇ 7 nAChR complexes are solubilized in 100 ⁇ l of SDS-PAGE buffer (62.5 mM Tris-HCl at pH 6.8). % glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.1% bromophenol blue) under heat for 5 minutes.
• the complexes are then deposited on an 8-16% SDS-polyacrylamide gel electrophoresis.
• Anti- ⁇ 7 nAChR monoclonal antibodies are used in the Western Blot analysis and then revealed by chemiluminescence. The intensity of the bands obtained is analyzed by densitometry in order to quantify the effects of the compounds as a function of their dose on the amount of A ⁇ 42 - ⁇ 7 nAChR complexes present.
• Compound S 24795 is used as an anti-amyloid agent in the ex vivo screening method protocol according to the invention.
• the compound S 24795 is a pyridine compound used as a mnemocognitive facilitator capable of improving the cognitive processes and / or of opposing the cognitive disorders associated with aging. In contrast to mnemocognitive facilitators acting directly on central cholinergic systems, compound S 24795 lacks hypothermic activity that may be troublesome in the treatment of patients with neurodegenerative disease. 1.8) Functional recovery method
• Functional recovery experiments are performed from 1.2-based brains from patients according to 1.1. Functional recovery induced by treatment with S 24795 (10 ⁇ M) is evaluated on the incoming calcium flux by the ⁇ 7 and NMDA receptors. It was tested after Ih treatment with S 24795 on control brains exposed to A ⁇ 42 (30 minutes) and on brains of patients with Alzheimer's disease. The treatments with A ⁇ 42 (1 ⁇ M) and S 24795 (10 ⁇ M) are performed on cortex slices from these brains. Synaptosomes are then prepared as described in 1.4. In order to evaluate the influx of Ca 2+ by the ⁇ 7 and NMDA receptors, the synaptosomes are incubated in the presence of 45 Ca 2+ (5 ⁇ M) for 5 minutes at 37 ° C.
• the screening method makes it possible to identify a preventive property of S 24795, added before the A ⁇ peptides, on the formation of the A ⁇ 42 - ⁇ 7 nAChR complexes.
• S 24795 added before the A ⁇ peptides
• FIG. 1 the addition of A ⁇ peptides to synaptosomes of non-diseased human cortex induces a sharp increase in the amount of A ⁇ 42 - ⁇ 7 nAChR complexes.
• the screening method makes it possible to identify a curative property of S 24795 since dissociating complexes formed before the death of the patient.
• S 24795 induces a major decrease in the amount of A ⁇ 42 - ⁇ 7 nAChR complexes present in diseased brains.
• the results illustrated in FIG. 3 confirm the ability of the screening method to identify a compound with a curative property in that, without the addition of A ⁇ peptides, this compound can induce dissociation of complexes already present in diseased human tissues.
• This screening method therefore makes it possible to specifically select compounds capable of preventing complex formation, which is applicable to early stages of the disease where the compounds have a preventive action, and secondly to dissociate already existing complexes, which is applicable to advanced or even severe stages of the disease, where the compounds have a curative action.
• the dissociation of the complexes A ⁇ 42 - ⁇ 7 nAChR will prevent the excessive intraneuronal accumulation of complexes A ⁇ 42 - ⁇ 7 nAChR and consequently oppose to the neuronal death due to these deposits.
• the realization of the screening method according to the invention from synaptosomes of human brains avoids the artifacts and false-positives related to the differences between the animal and human species.
• the ex vivo implementation of this screening method makes it possible to obtain a speed and a repeatability for the identification of compounds capable of to dissociate ⁇ -amyloid peptide complexes with nicotinic acetylcholine receptors.
• this screening method is carried out on biological material representing a severe and fixed stage of Alzheimer's disease, it makes it possible to select and identify compounds acting at an ultimate stage of the disease.
• the screening method according to the invention ensures the identification of compounds that can be used in the curative treatment of neurodegenerative diseases and of Alzheimer's disease in particular.

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• 2006
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