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WO2008142194A1 - Méthode pour le diagnostic/pronostic in vitro de la chorée de huntington - Google Patents

Méthode pour le diagnostic/pronostic in vitro de la chorée de huntington Download PDF

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Publication number
WO2008142194A1
WO2008142194A1 PCT/ES2008/070092 ES2008070092W WO2008142194A1 WO 2008142194 A1 WO2008142194 A1 WO 2008142194A1 ES 2008070092 W ES2008070092 W ES 2008070092W WO 2008142194 A1 WO2008142194 A1 WO 2008142194A1
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Prior art keywords
huntington
chorea
neurons
antagonist
mice
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Spanish (es)
Inventor
José Javier LUCAS LOZANO
María Teresa MIRAS PORTUGAL
Miguel DÍAZ HERNÁNDEZ
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Complutense de Madrid
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Complutense de Madrid
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention can be encompassed within the field of neurodegeneration and genetics, specifically within the field of neurological diseases caused by genetic-based alterations, specifically Huntington's chorea.
  • the present invention relates to a method of diagnosis and / or prognosis, in vitro, of Huntington's chorea based on the expression and / or activity of the P2X7 gene at the neuronal level, to a kit comprising at least capable gene sequences of hybridizing with sequences belonging to the P2X7 gene for the diagnosis and / or prognosis, in vitro, of Huntington's chorea.
  • antagonists capable of crossing the blood-brain barrier which are blockers of the activity of the P2X7 receptor, and pharmaceutically acceptable vehicles, for the preparation of drugs and pharmaceutical compositions, intended for the treatment of Huntington's chorea, is described.
  • Also included in the present invention are methods of detecting drugs for Huntington's chorea and methods of identifying effective doses of P2X7 inhibitors, based on the neuronal alterations associated with P2X7.
  • neuro image probes are described that allow visualizing the increments of neuronal P2X7 associated with Huntington's chorea, and methods of obtaining neuroimaging using said probes.
  • HD Huntington's disease
  • Huntington's disease is one of the most representative of neurodegenerative diseases due to the expansion of a CAG triplet in the DNA.
  • This triplet encodes the amino acid glutamine and the result is that, in adulthood, the encoded proteins can contain up to hundreds of consecutive glutamine residues.
  • These zones of polyglutamine (polyQ) interfere with the functions of the native protein or cause the appearance of a new toxic function. This type of anomalies is aggravated in successive generations, as the expansion of the CAG triplet increases.
  • Huntington's disease has a genetic basis where the gene with polyglutamine expansion is the one that encodes the abnormal huntingtin protein.
  • the gene called IT15 is near the end of the short arm of chromosome 4 (4pl6.3).
  • Huntington's disease there can be from 37 to 240 repetitions of the CAG triplet, which encodes as many glutamines. In healthy individuals there can be between 11 and 35 repetitions of this triplet without any anomaly. From 42 repetitions, the disease appears with all its symptoms and its development is proportional to the length of the polyglutamine (poly-Q) chain.
  • This expansion of polyglutamines is close to the N-terminal end of the protein, altering its function, its turnover and forming cytosolic and nuclear clusters.
  • This protein is It expresses abundantly in the cerebral cortex and the basal ganglia, which are essential areas for movement control. The first symptoms appear at the end of the second decade of life and the severity of the symptoms depends on the expansion of the CAG triplet.
  • the direct involvement of huntingtin in the genesis of the disease has been proven experimentally in mice whose brain has been genetically modified to produce the elongated version of the protein, these rodents presenting brain damage and mobility problems very similar to those observed in the patients.
  • GABAergic striatum terminals have a high presence of nucleotide (P2X) ionic tropic receptors and dinucleotides. These presynaptic receptors that allow the massive entry of the calcium ion, are in turn modulated by the receptors for the GABA itself, of the GABA B metabotropic type, which are co-located in the same terminals.
  • P2X nucleotide
  • P2X7 receptors are cationic channels powered by ATP, which are known to modulate the release of neuro transmitters from presynaptic terminals.
  • the P2X7 receptor was initially detected in rat brain and is abundantly expressed in cells of the hematopoietic lineage and in multiple cell types of the immune system. Subsequently, it was detected in microglia and neurons, especially in synaptic terminals. The stimulation of P2X7 in the immune system regulates the production and release of cytokines.
  • US2005 / 0288288 refers to piperidine derivatives that act as antagonists of the P2X7 receptor and are useful in the treatment of inflammatory, immunological or cardiovascular diseases.
  • the efficacy of an inhibitor would be greater or only work if it is able to cross the blood brain barrier, so that it can interact directly with P2X7 receptors at the level of the central nervous system.
  • P2X7 receptor antagonists it is not necessary that they be delivered directly to the central nervous system, but by other routes that are easier to access and more effective, such as the intraperitoneal and intravenous routes.
  • the present invention concretely demonstrates that BBG is more effective than the rest of the P2X7 antagonists described in the prior art since it is capable of crossing the blood brain barrier, demonstrating that BBG administered intraperitoneally to mice reaches the cerebral parenchyma at concentrations compatible with an antagonistic effect on the P2X7 receptor, that the BBG administered intraperitoneally to R6 / 1 mice stops the progressive deterioration of motor coordination in the rota-rod test, and that BBG administered Intraperitoneally, R6 / 1 mice prevent neural death, since caspase-3 positive (apoptotic) cells decrease in the cortex.
  • the administration of BBG produces fewer adverse side effects because the antagonist directly targets the P2X7 receptor of neurons, since it is only in the models in which the mutated htt is present in which it works.
  • the present invention relates specifically to a method of diagnosis and / or prognosis, in vitro, of Huntington's chorea based on the expression and / or activity of the P2X7 gene and the use of antagonists, capable of crossing the blood brain barrier. which are blockers of the activity of the P2X7 receptor, and pharmaceutically acceptable vehicles, for the preparation of drugs intended for the treatment of Huntington's chorea.
  • a kit is described which comprises at least gene sequences capable of hybridizing with sequences belonging to the P2X7 gene for diagnosis and / or prognosis, in vitro, of Huntington's chorea.
  • the development of the present invention was carried out by observing the expression of mutated huntingtin in the areas of the cortex and / or the striatum of the brain and peripheral blood (SP) in animal models, as well as in neurons in culture.
  • SP peripheral blood
  • the third model consists of primary cultures of wild mouse cortical and striatal neurons that are transfected with expression plasmids encoding the fragment N-terminal huntingtin with a poly-Q expansion of 17 glutamines, which behaves as normal, and with other expansions of 72 or 84 glutamines that reproduce aspects of Huntington's disease. These fragments are attached to GFP (Green Fluorescent Protein) or CFP (Cyan Fluorescent Protein) for easy viewing.
  • GFP Green Fluorescent Protein
  • CFP Cyan Fluorescent Protein
  • neurons and synaptosomes were loaded with a FURA-2AM solution (5 ⁇ M) for 30 min. at 37 0 C, to allow intracellular hydrolysis of the FURA-2AM dye.
  • FURA-2AM solution 5 ⁇ M
  • the increases in calcium induced in neurons or nerve terminals were analyzed as they were stimulated with selective P2X7 receptor agonists.
  • the coverslips where the neurons or synaptosomes were stuck with polylysine were mounted in a superfusion chamber in a Nikon Eclipse TE-2000 microscope.
  • the test medium was HBM (140 mM NaCl, 5 mM KCl, 1.2 mM NaH 2 PO 4 , 10 mM glucose and 10 mM HEPES, pH 7.4).
  • the neurons were superfused at a rate of 1.2 ml / min with perfusion medium free of Mg 2+ during functional tests. Initially, 10 ⁇ M Bz-ATP pulses were applied to the neurons for 30 seconds, in the absence of any other drug. Then, the neurons were incubated for 5 min. with BBG (1 ⁇ M) and retested with 10 ⁇ M Bz-ATP in the presence of the antagonist. A pulse of K + 30 mM was finally applied at the end of each experiment to confirm the viability of the neurons and the functionality of the synaptosomes studied. All compounds were dissolved in HBM medium free of Mg 2+ . The neurons were visualized using a Nikon microscope with a 0.5-1.3 S Fluor 40 magnification oil lens.
  • the wavelength of the incoming light was filtered to 340 nm and 380 nm with the aid of a monochromator (10 nm bandwidth, Optoscan moncromator, Cairin).
  • the images were obtained with a CMA camera ORCA-ER C 47 42-98 from Hamamatsu controlled by Metafluor 6.3r6 software (Universal Imaging Corp., Cambridge, UK).
  • the exposure time was 250 ms for each wavelength and the change time ⁇ 5 ms.
  • the images were taken continuously and stored on a fast SCSI disk. Data over time represents the average light intensity in a small elliptical region within each cell.
  • the background and autofluorescence components were subtracted for each wavelength. According to the results obtained in Fig.
  • the percentage of synaptosomes that responded to the antagonist was significantly higher in the preparation of Tet / HD94 mice. Records of calcium images in response to exposure to BzATP produced two types of kinetics. In one case, the synaptosomes showed an initial peak that remained elevated during exposure to BzATP, and decreased progressively during the washing of the BzATP (decreasing profile). The second profile was characterized by multiple fluorescence peaks in the increasing sense of the scale, which remained for at least 25 seconds after the elimination of BzATP (increasing profile). In synaptosomal preparations of wild mice both profiles were similarly represented. However, in Tet / HD94 synaptosomal preparations, the percentage deviated in favor of the growing profile.
  • Live neurons were detected with calcein AM (acatoxymethyl ester) and cell death with propidium iodide, which crosses the membrane of dead cells to mark their DNA.
  • D compares cultures of cortical and striatal neurons of wild mouse and Tet / HD94 model, appreciating how BzATP, which is harmless in cultures of wild mice, increases cell death in cortical neurons of the Huntingtonian model, an effect that It is reversed with the use of a P2X7 antagonist such as BBG.
  • Cell death can also be detected by other equivalent techniques such as: TUNNEL staining, detection of activated caspases, detection of LDH, etc.
  • the findings cited in the present invention may constitute an important tool for the diagnosis, prognosis and cure of Hungtington's chorea, as the results cited below are clear from them.
  • P2X7 is proposed as a marker for asymptomatic patients in which an increase in mRNA or protein may indicate the proximity of the onset of Huntingtonian symptomatology, as well as the progression of the disease per se and in response to a possible treatment.
  • the present invention proposes the use of P2X7 receptor antagonists or inhibitors, such as BBG (Brilliant Blue G) or any other that can cross the blood-brain barrier and have a selective action on the neuronal P2X7, for the preparation of a pharmaceutical composition for the treatment of Huntington's chorea, by attenuating neuronal alterations (changes in calcium entry and increased vulnerability to apoptosis) produced as a consequence said disease and resulting from the expression of mutated huntingtin in the affected neurons themselves.
  • BBG Brown Blue G
  • an in vitro model of identification of drugs that enhance the protective effect on neurons treated with P2X7 inhibitors and transfected with the N-terminal region of exon 1 of the htt-72Q was developed, measuring specific responses in the terminals of the neurons in culture.
  • the constitution, through neuron cultures, of the transgenic or transfected models with mutated huntingtin, of an in vitro drug identification system that, co-administered with P2X7 inhibitors, reduces the alterations in Calcium entry or vulnerability to neuronal death, and dose identification of P2X7 inhibitors have beneficial effects on neurons that express mutated htt (these doses would have a lower risk of adverse effects).
  • the first aspect of the present invention relates to an in vitro diagnostic and / or prognosis method of Huntington's chorea, based on the expression and / or activity of the P2X7 gene, which can be performed both at the level of mRNA as of protein.
  • the consequence of the increase in the transcription of P2X7 in the cerebral cortex, is a noticeable increase of the protein in the striated area, located by the Western type transfer technique (WB).
  • WB Western type transfer technique
  • it was found that the increase in P2X7 receptor is also detected in peripheral blood of R6 / 1 mice, in an amount 2.5 times higher than the control (by WB).
  • the diagnostic / prognostic method can be carried out in both cerebrospinal fluid and peripheral tissues (specifically peripheral blood) using the RT-PCR technique or with antibodies.
  • Sampling can be done both at the level of the cerebrospinal fluid and at the level of the peripheral blood.
  • the increase in P2X7 can be analyzed in the previous samples at the mRNA level or at the protein level.
  • the proportion of P2X7 messenger RNA with respect to an internal control is determined by the RT-PCR technique or others that serve the same purpose.
  • the proportion of P2X7 protein with respect to an internal control is determined by the WB technique with antibodies specific for P2X7 and for the respective internal control. On the same basis an ELISA detection could be established.
  • Another way of detecting the increase of P2X7 at the protein level in the aforementioned samples is by flow cytometry using a P2X7 ligand detectable by luminometric or fluorimetric techniques (such as BBG itself) and determining the proportion of this marker with respect to signal in flow cytometry of an internal control proportional to the number of cells or simply with respect to the number of cells detected by the cytometer
  • a second aspect of the present invention relates to a kit comprising at least gene sequences capable of hybridizing with sequences belonging to the P2X7 gene for the diagnosis and / or prognosis, in vitro, of Huntington's chorea.
  • the third aspect of the present invention relates to the use of antagonists capable of crossing the blood brain barrier, which are blockers of the activity of the P2X7 receptor, and of pharmaceutically acceptable vehicles, for the preparation of medicaments for the treatment of Huntington's chorea.
  • said antagonist may be BBG, KN-62 or decavanadate and where said antagonists may have a ligand or probe detectable attached at the brain level by neuro image techniques.
  • the fourth aspect of the present invention relates to pharmaceutical compositions comprising antagonists capable of crossing the blood brain barrier, which are blockers of P2X7 receptor activity, and pharmaceutically acceptable carriers, intended for the treatment of Huntington's chorea in which said Compositions may comprise probes or ligands detectable at the brain level by neuro imaging techniques.
  • the fifth aspect of the present invention relates to a method of in vitro detection of drugs useful for the treatment of Huntington's chorea, which decrease the neuronal alterations induced by P2X7 activating substances (eg agonists), which comprises the following steps: a) measure the concentration of calcium in the terminals and / or the proportion of apoptosis in cultured neurons that express the mutated huntingtin protein, in the presence and absence of the drug evaluated; b) calculate the difference between both concentrations; c) consider those drugs in which said difference is statistically significant (p ⁇ 0.05).
  • P2X7 activating substances eg agonists
  • the detection of apoptosis can be performed by any usual method, eg staining with propidium iodide and calcein.
  • the last aspect of the invention presents a method of identifying effective doses of P2X7 inhibitors for the treatment of Huntington's chorea, which comprises the following steps: a) measuring the concentration of internal calcium and / or the proportion of apoptosis in neurons in culture expressing mutated huntingtin, using these measures as control; b) in parallel, administering a dose of the inhibitor to a culture of neurons similar to that of step a); c) measure the calcium concentration and the proportion of neuronal apoptosis in the culture of stage b); d) identify the doses that produce a significantly lower calcium concentration and apoptosis ratio (p ⁇ 0.05) than controls a).
  • cultures of neurons of the transgenic models or of wild animals transfected with mutated huntingtin or cultures of any cell line such as neuroblastomas, pheochromocytomas or others with neuronal properties and transfected with mutated forms of htt can be used.
  • neuroimaging probes were generated in the present invention to detect said increase in the brain of HD patients, allowing to assess the state of disease progression and the possible efficacy of a therapy. Therefore, it is the generation of a product, such as a P2X7 receptor ligand, which is detectable by neuroimaging techniques, such as [HC] raclopride.
  • the seventh aspect of the present invention relates to probes designed to be detected by neuroimaging techniques that allow the identification of the increase in transcription and / or concentrations of P2X7 at the patient's brain level.
  • the last aspect of the invention relates to the use of said probe in a method of obtaining neuro images, which would allow analyzing the progression of the alteration of P2X7 concentrations as an indicator of the stage of disease progression in a specific patient. over time, or as an indicator of the possible efficacy of a treatment after different times, doses, or routes of treatment administration.
  • the present figure shows the increase in P2X7 levels in the striatum and blood of mouse models of Huntington's disease.
  • Immunoblot analysis of brain (left) and striatal (right) cortex extracts of two different mouse models of Huntington's disease (conditional model Tet / HD94 (black bars) and R6 / 1 (gray bars)) is represented, and control animals (white bars) with antibodies against P2X7.
  • the membranes were incubated with anti- ⁇ tubulin ( ⁇ -Tub) to correct any possible deviation in the amount of protein.
  • Histograms show the metric densified quantification of P2X7 levels in both models of transgenic mice versus controls at different ages (3 to 16 months for Tet / HD94, and 3 to 10 months for R6 / 1).
  • the levels of cortical and striatal P2X7 messenger RNA were measured in 16-month-old Tet / HD94 mice and in 10-month R6 / 1 mice, and in their corresponding controls at the same age. Histograms show the quantification of the P2X7 messenger in both models versus controls.
  • the levels of P2X7 protein in peripheral blood of R6 / 1 mice were also quantified, observing a 2.5-fold increase compared to controls (* p ⁇ 0.01, ** p ⁇ 0.005, *** P ⁇ 0.001). It is noteworthy that in peripheral blood the increase in expression levels in the Huntingtonian mouse model is 2.5 times average with respect to the control.
  • WT Western transfer.
  • a and B 16-month-old cortical (A) and striatal (B) sections of Tet / HD94 are represented with anti-P2X7.
  • C and D A triple immuno fluorescence is represented with anti-P2X7 and anti- ⁇ lll tubulin (BIII Tub) antibodies and Dapi nuclear marking of cortical sections of 16-month-old Tet / HD94 mice.
  • the empty arrows show apoptotic nuclei of pyramidal neurons that express P2X7.
  • White arrows indicate cortical projections that express P2X7.
  • E A 10-month R6 / 1 mouse brain sagittal section depicting P2X7 at cortical level (Cx), corpus callosum (CC) and striatum (St).
  • a double immuno fluorescence is represented with anti-P2X7 and anti-DARPP-32 (DARPP-32) of R6 / 1 mouse striatum of 10 months of age. Scales: in A and B 100 ⁇ m; C and D 25 ⁇ m.
  • the present figure represents the increased expression and altered response of P2X7 in synaptic terminals of the anterior brain of Tet / HD94 mice.
  • a and B It represents a double immuno fluorescence of synaptic anterior brain terminals marked with anti-synaptophysin and anti-P2X7 control animals (A) and Tet / HD94 (B).
  • C Represents a histogram showing the quantification of synaptic terminals positive for P2X7 in control mice (white bar) and Tet / HD94 (black bar).
  • the present figure shows the altered calcium permeability in which P2X7 intervenes, in terminals and somas of primary neurons that express mutant htt.
  • Cultures of primary bark neurons were prepared from wild mice, which express the N-terminal fragment of the htt with the normal polyQ fragment (17 CAG), and mutants, which express the expanded fragment (72 CAGs), fused to the GFP .
  • A Image of a cortical neuron that expresses the fragment of 72 CAGs fused to the GFP (N-htt72QGFP).
  • the empty arrows show nuclear accumulation of fluorescence in the soma, which corresponds to aggregates of the polyQ fragment of the htt, and the arrows filled with aggregates in the neuropil.
  • B It shows the same field of cortical neurons as in image A revealed with the Fura-2 probe. In both figures the triangular arrows show the presence of N-htt72QGFP in terminals of transfected neurons, the circular arrows indicate nerve terminals of non-transfected neurons.
  • C Fluorescence means (Fura-2) over time of two types of response of registered nerve terminals, of transfected and non-transfected cortical neurons stimulated with 10 ⁇ M BzATP. Also shown are the percentages of synaptic terminals that show each type of kinetic profile in response to Bz-ATP, both in transfected and non-transfected neurons. (CONT: Control).
  • D Changes in fluorescence (Fura-2) over time, recorded in soma of cortical neurons not transfected or transfected with N-httl7QGFP or N-htt72QGFP, in response to 0.5 ⁇ M and 10 ⁇ M of Bz-ATP.
  • Histograms show the quantification of untransfected cortical neurons (white bar), transfected with the 17 repetition fragment (gray bar) and with the 72 repetition fragment (black bar), with somas that respond to both doses of BzATP.
  • E The increase in intracellular calcium induced by 10 ⁇ M BzATP in cortical neurons was suppressed when the neurons were previously incubated with 1 ⁇ M BBG for 2 minutes. The bars indicate the periods of stimulation with the components tested in the different experiments. All analyzed neurons responded to stimulation with K + 30 mM.
  • the present figure represents the increase in cell death in cortical neurons of Tet / HD94 mice in response to treatment with BzATP.
  • Primary cultures of cortical neurons of control mice and Tet / HD94 were treated with BzATP 10 ⁇ M for 2 hours and analyzed 48 hours later.
  • a and B Fluorescence images of cortical neurons of control (A) and Tet / HD94 (B) mice labeled with calbindin and propidium iodide after treatment with BzATP. In some cases, cortical neurons were previously treated with 1 ⁇ M BBG 10 minutes before stimulation with BzATP.
  • C Histograms show the quantification of cortical (left) and striatal (right) neurons positively labeled for the anti-calbindin antibody after treatment with BzATP in the presence or absence of BBG. *** p ⁇ 0.001.
  • the present figure shows the in vivo treatment of R6 / 1 huntingtonian mice with BBG.
  • Wild mice (WT) and R6 / 1 were treated with BBG (1 mg / 22 mg body weight) or with saline solution every 48 hours for 21 days.
  • BBG concentrations were measured in both blood (left) and brain (right) of treated animals, with levels of 2.25 ⁇ m and 13OnM observed. (UA: Absorbance units).
  • B. The upper graph shows the residence time of the mice in the rota- rod, showing a significant brake in the progression of the disease in R6 / 1 mice treated with BBG.
  • the graph below shows the evolution in the body weight of the animals in the experiment. A smaller drop in the weight of R6 / 1 mice treated with BBG than in those treated with saline alone is observed.
  • the present figure shows the reduction in the incidence of cell death in the cortex of nine-month-old R6 / 1 mice in response to treatment with BBG in vivo. After treatment, mice were sacrificed and the incidence of positively labeled neurons for the endoproteolized anti-caspase-3 antibody in cortex and striatum was analyzed.
  • AB Cortical sections of R6 / 1 mice treated with saline (A) or BBG (B) labeled with endoproteolyzed anti-caspase-3 antibodies.
  • the arrows indicate the presence of marked neurons, with apoptotic morphology.
  • the tables show a 2.Ox increase in apoptotic cells identified with the arrows.
  • CD Striatal sections of R6 / 1 mice treated with saline (C) or BBG (D) labeled with endoproteolyzed anti-caspase-3 antibodies.
  • the arrows indicate the presence of marked neurons, with apoptotic morphology.
  • E-F Histograms show the quantifications of positively labeled cells with anti-caspase-3 antibodies in the cortex and striatum of wild mice (white bars) or R6 / 1 (black bars) treated with or without BBG. * p ⁇ 0.01.
  • WT Western transfer.
  • EXAMPLE 1 Increased levels of P2X7 in the striatum and blood of mouse models of Huntington's disease.
  • the concentrations of cortical and striatal P2X7 messenger RNA were measured in 16-month-old Tet / HD94 mice and in 10-month R6 / 1 mice, and in their corresponding controls at the same age. Histograms show the quantification of the P2X7 messenger in both models versus controls. The levels of P2X7 protein in peripheral blood of R6 / 1 mice were also quantified, observing a 2.5-fold increase compared to controls (* p ⁇ 0.01, ** p ⁇ 0.005, *** P ⁇ 0.001). The present example highlights that in peripheral blood the increase in expression levels in the transgenic mouse model is 2.5 times compared to the control. EXAMPLE 2. Location of P2X7 in cortico-striatal projections.
  • EXAMPLE 3 Increased expression and altered response of P2X7 in synaptic terminals of the anterior brain of Tet / HD94 mice.
  • the present example shows the cell death of cortical neurons of Tet / HD94 mice due to treatment with BzATP.
  • Primary cultures of cortical neurons of control mice and Tet / HD94 were treated with BzATP 10 ⁇ M for 2 hours and analyzed 48 hours later (Figure 5). Fluorescence images of cortical neurons were obtained from control mice and Tet / HD94 labeled with calbindin and propidium iodide after treatment with BzATP. In some cases, cortical neurons were previously treated with 1 ⁇ M BBG 10 minutes before stimulation with BzATP. Histograms were performed showing the quantification of positively labeled cortical and striatal neurons for the anti-calbindin antibody after treatment with BzATP in the presence or absence of BBG. *** p ⁇ 0.001.
  • Wild mice (WT) and R6 / 1 were treated with BBG (1 mg / 22 mg body weight, corresponding to 45.5 mg / kg body weight) or with saline solution every 48 hours for 21 days (Figure 6).
  • BBG levels, both in blood and in brain, of the treated animals were measured, observing levels of 2.25 ⁇ m and 13OnM.
  • B. The residence time of the mice in the rota-rod was shown, with a significant brake on the progression of the disease in the R6 / 1 mice treated with BBG and the evolution in the body weight of the animals in the experiment. A smaller drop in the weight of R6 / 1 mice treated with BBG than in those treated with saline alone was observed.
  • EXAMPLE 6 Treatment with BBG in vivo reduces the incidence of cell death in cortex of R6 / 1 mice aged nine months.
  • mice After treatment with BBG, the mice were sacrificed and the incidence of positively labeled neurons for the endoproteolized anti-caspase-3 antibody in cortex and striatum was analyzed (Figure 7). Neurons were immunohistochemically detected apoptotic marked with anti-caspase-3. Cortical sections of R6 / 1 mice treated with saline solution or with BBG labeled with digested anti-caspase-3 antibodies were visualized, locating the presence of labeled neurons, with apoptotic morphology. Striatal sections of R6 / 1 mice treated with saline or BBG solution labeled with digested anti-caspase-3 antibodies were performed and the presence of labeled neurons was indicated, with apoptotic morphology. Histograms were performed showing the quantifications of positively labeled cells with anti-caspase-3 antibodies in cortex and striatum of wild or R6 / 1 mice treated with or without BBG. * p ⁇ 0.01.

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Abstract

L'invention concerne une méthode de diagnostic/pronostic in vitro de la chorée de Huntington, qui est fondée sur l'augmentation de l'expression et/ou de l'activité du gène P2X7 au niveau neuronal et sur l'utilisation d'antagonistes de P2X7, qui peuvent traverser la barrière hémato-encéphalique, et des excipients acceptables sur le plan pharmaceutique pour l'élaboration de médicaments et de compositions pharmaceutiques à utiliser pour le traitement de la chorée de Huntington. L'invention concerne également un kit diagnostique qui comprend des séquences géniques pouvant s'hybrider avec des séquences appartenant au gène P2X7. L'invention concerne en outre des méthodes de détection d'agents pharmaceutiques et d'identification de doses efficaces d'inhibiteurs. Enfin, l'invention concerne des sondes et des méthodes de neuroimagerie qui permettent de visualiser ladite augmentation de l'activité de P2X7.
PCT/ES2008/070092 2007-05-17 2008-05-14 Méthode pour le diagnostic/pronostic in vitro de la chorée de huntington Ceased WO2008142194A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011109833A2 (fr) 2010-03-05 2011-09-09 President And Fellows Of Harvard College Compositions de cellules dendritiques induites et utilisations associées

Citations (3)

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Publication number Priority date Publication date Assignee Title
US6133434A (en) * 1997-04-28 2000-10-17 Glaxo Group Limited Purinergic receptor
WO2005041892A2 (fr) * 2003-11-03 2005-05-12 Cornell Research Foundation, Inc Inhibition du recepteur des purines en tant que strategie therapeutique au niveau de la moelle epiniere et du cerveau
US20050288288A1 (en) 2004-06-29 2005-12-29 Pfizer Inc. Methods for preparing P2X7 inhibitors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6133434A (en) * 1997-04-28 2000-10-17 Glaxo Group Limited Purinergic receptor
WO2005041892A2 (fr) * 2003-11-03 2005-05-12 Cornell Research Foundation, Inc Inhibition du recepteur des purines en tant que strategie therapeutique au niveau de la moelle epiniere et du cerveau
US20050288288A1 (en) 2004-06-29 2005-12-29 Pfizer Inc. Methods for preparing P2X7 inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LE FEUVRE R. ET AL.: "Extracellular ATP and P2X7 receptors in neurodegeneration", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 447, 2002, pages 261 - 269, XP003024148 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011109833A2 (fr) 2010-03-05 2011-09-09 President And Fellows Of Harvard College Compositions de cellules dendritiques induites et utilisations associées

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