WO2008142194A1

WO2008142194A1 - Method for the in vitro diagnosis/prognosis of huntington's chorea

Info

Publication number: WO2008142194A1
Application number: PCT/ES2008/070092
Authority: WO
Inventors: José Javier LUCAS LOZANO; María Teresa MIRAS PORTUGAL; Miguel DÍAZ HERNÁNDEZ
Original assignee: Universidad Complutense De Madrid; Consejo Superior De Investigaciones Científicas
Priority date: 2007-05-17
Filing date: 2008-05-14
Publication date: 2008-11-27

Abstract

The invention relates to a method for the in vitro diagnosis/prognosis of Huntington's chorea, based on an increase in the expression and/or activity of the P2X7 gene at neuronal level, to the use of P2X7 antagonists capable of penetrating the hematoencephalic barrier and to pharmaceutically acceptable vehicles for the production of pharmaceutical compositions and drugs intended for the treatment of Huntington's chorea. The invention also relates to a diagnostic kit comprising gene sequences capable of hybridising with sequences belonging to the P2X7 gene. The invention further relates to methods for detecting drugs and for identifying effective doses of inhibitors. Furthermore, the invention relates to neuroimaging methods and probes that can be used to view the above-mentioned increase in the activity of P2X7.

Description

IN VITRO DE LA KOREA DIAGNOSTIC / FORECAST METHOD

HUNTINGTON

FIELD OF THE INVENTION

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. Specifically, 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. Likewise, 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 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.

Finally, 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.

STATE OF THE TECHNIQUE

Huntington's disease (HD) is a neurological, degenerative, hereditary, autosomal dominant disease whose three most important manifestations are: the appearance of uncontrolled movements, called chorea, with loss of intellectual abilities and behavioral changes. They were first described by Dr. George Huntington in 1872. Therefore, HD is a disease that affects the brain, specifically the areas of the cortex and / or the striatum, where neurons progressively degenerate.

Huntington's disease (HD) 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.

Its incidence is approximately 10 patients per 100,000 inhabitants, which represents a relatively high frequency, in addition it is transmitted in an autosomal dominant way, with fatal development from 35-40 years. Currently, despite the existence of medications that can alleviate some of the symptoms associated with HD, there is no known treatment that is really effective for the cure of the disease. In addition, the effectiveness of these varies depending on the patient and tend to be less effective as the disease progresses.

As previously mentioned, and is arranged in the state of the art, 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). In 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.

There seems to be a significant loss of GABAergic striated neurons, which would explain the loss of GABA-mediated inhibitory modulation in the basal ganglia. Also from the early stages there is a generalized atrophy of the cerebral cortex and throughout the progression of the disease patients present with loss of neurons from the cerebral cortex. In more advanced stages of the disease, cholinergic and dopaminergic neurons also decrease. The death of striatum neurons in patients affected by Huntington's disease caused an activating effect of the mutated protein on caspases that participate in neuronal apoptosis. 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.

Additionally, it has recently been established that extracellular ATP causes neuronal death through stimulation of P2X7 receptors. These 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. This document also cites the possible application of these antagonists in the treatment of neurodegenerative diseases, specifically Huntington's disease. But the connection between P2X7 and Huntington's chorea in US 2005/0288288 derives from the role of P2X7 in the inflammation and intermediation of interleukins such as IL-1 through the glia and, instead, in the present invention. it is demonstrated that the altered receptor in its concentrations and structure is the neuronal P2X7 receptor and that it is in the damaged areas of the central nervous system where the elevated concentrations thereof are found. Specifically, it is in the terminals of cortical neurons where the high concentration of P2X7 increases the Ca ⁺⁺ transit and induces a greater response, without the need for intermediation of interleukins or any other cytokine or the participation of cells of the immune system to that neuronal death occurs, as demonstrated in pure neuronal cultures without the presence of other cell types during the development of the present invention. Therefore, although an administration of P2X7 inhibitors produced a decrease in IL-I in extracerebral cells of the immune system, and this led to an improvement in the disease, according to the present invention, inhibition of neuronal P2X7 receptors could attenuate alterations in calcium entry and vulnerability to apoptosis of affected neurons in HD. Therefore, in the case of the present invention, 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. This demonstrates that to alleviate the symptoms of Huntington's chorea using 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. In addition, 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.

Although the experiments of the present invention were performed with BBG, the experimental data constitute clear evidence that any other P2X7 inhibitor that crosses the blood brain barrier would produce similar effects. Other examples of such inhibitors would be decavanadate ([Vioθ2δ] 6-) or KN-62 (l- [N, O-bis- (5- isoquinolin-sulfonyl) -N-methyl-L-tyrosyl] -4-phenyl -piperazine)

DESCRIPTION OF THE INVENTION

Brief Description of the Invention

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. On the other hand, in the present invention 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. Detailed description of the invention

The hypothesis was raised that the alteration of calcium permeability mediated by P2X7 could contribute to the synaptic dysfunction and increased apoptosis observed in Huntington's chorea (HD). Using cell and HD mouse models, increased concentrations of the receptor and altered P2X7-mediated calcium permeability in HD neuron terminals and terminals were demonstrated. In addition, cultured neurons expressing mutated huntingtin showed an increased susceptibility to apoptosis triggered by stimulation of the P2X7 receptor.

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.

Initially, the presence of the P2X7 receptor and other markers in the areas mainly affected in Huntington's disease, such as the cortex and the striatum, was studied using R6 / 1 mouse models (one of the first transgenic mice designed to study this disease) expressing mutated huntingtin ubiquitously, where the expression of the transgene is directed by the huntingtin promoter. In addition, the study of the presence of the P2X7 receptor in peripheral blood was also carried out (Fig. 1)

Subsequently, the brain areas for the presence of the P2X7 receptor and others were studied, using peripheral blood as a control, in models of Tet / HD94 mice (also called HD94) that express huntingtin conditionally, by controlling tetracycline in the promoter, where the poly-Q tail has 94 glutamines. Neural preparations of this model were also used for response studies in synaptic terminals, demonstrating the unequivocal presence of the P2X7 receptor with presynaptic location (Fig. 2)

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. In these transfected neurons, the presence of the P2X7 receptor with different properties is demonstrated depending on whether we have the normal or expanded fragment of the mutated htt, where the Ca ⁺⁺ input is much greater. The psycho-chemical properties of the receptor are altered, observing greater affinity and greater response to both the physiological ligand and the pharmacological ligand. This response can be inhibited by the BBG inhibitor.

From these findings, referring to the expression of mutated huntingtin, in the present invention the occurrence of the events explained below was checked.

In the models of transgenic mice of Huntington's disease it was observed, using the quantitative RT-PCR technique, a very significant increase in the transcription of P2X7 in the cerebral cortex of these animals with respect to the control mice. As a consequence of the increase in the transcription of P2X7 in the cerebral cortex, a noticeable increase of the protein in the striatum zone is observed, using the Western-type transfer technique (WB). In addition, the neural location is confirmed, since P2X7 in immuno-histochemical techniques is observed to be located in the axons that cross the corpus callosum and that contact the striatal location neurons. On micrographs, the abundant presence of P2X7 is observed in the axons leaving the cortex and its joint location with specific axon markers (beta-3 microtubulin, exclusively axonal marker). They cross the corpus callosum to form buttons around the striatal neurons positive for the DARPP-32 marker specific to striatal projection neurons (the ones that most notably degenerate into HD). It was observed how convergence occurs around the neural somas without the joint location of both markers. Therefore, it can be concluded that P2X7 is axonal and marks the terminals around striatal neurons. It was also seen, through the WB technique, 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.

On the other hand, alteration was detected in the physiology of the P2X7 receptor, both in affinity and in the maximum response. In neurons that express mutated htt, the receptors are able to respond to lower concentrations of the agonist, producing changes in the pattern of calcium entry in response to P2X7 agonists in synaptosomes of the animal model Tet / HD94 and in wild neurons transfected with huntingtin mutated (Fig. 3).

Next, calcium permeability was analyzed in isolated nerve terminals as well as in cortical neurons in culture transfected or not with the expanded polyQ fragment, in response to a 30-second exposure to a 10 μM concentration of the P2X7 BzATP agonist (Fig. . 4). In the case of isolated nerve terminals or synaptosomes, the percentage of them that responded to the agonist was significantly higher in those that came from transgenic Huntingtonian mice (Tet / HD94) than in those from control mice. Likewise, analyzes were performed micro fluorimetric calcium concentration. Similar results were obtained when the nerve terminals of neurons maintained in culture for three days and transfected with the mutated htt were analyzed. To perform these tests, neurons and synaptosomes were loaded with a FURA-2AM solution (5 μM) for 30 min. at 37 ⁰ C, to allow intracellular hydrolysis of the FURA-2AM dye. Using this probe, 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 µM 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 μM) and retested with 10 μM 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. 3, 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. These data suggest that, in addition to the increased concentrations of P2X7 receptor in the striatum of HD mice, there is an alteration of the functional state of the receptor, at the level of the synaptic terminal. On the other hand, when neurons were pre-incubated with 1 μM BBG, the increase in intracellular calcium induced by 10 μM BzATP was suppressed. In addition, it was shown that treatment with BzATP (P2X7 agonist), at doses that do not affect the viability of neuronal cultures of wild mice, increases cell death in cortical neurons of Tet / HD94 mice. Primary cultures of cortical neurons of control mice and Tet / HD94 were treated with BzATP 10 μM for 2 hours and analyzed 48 hours later. 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. In fig. 5-C, 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.

It was observed, in the model animals in which the expression of htt is ubiquitous, that throughout the progression of the disease and before it is symptomatic, the concentrations of mRNA and P2X7 protein in peripheral blood are markedly increased. For this reason, in the present invention 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.

In addition, 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. In models in culture it was seen that BBG neutralizes the excito-toxic actions of BzATP in neurons. Likewise, it was found that treatment with BBG injected intraperitoneally in vivo reduces the incidence of cell death in cortex of R6 / 1 mice of nine months of age. After the sacrifice of the mice, the endoproteolized caspase-3 (as a marker of cell death) in cortex and striatum was analyzed, showing that BBG treatment significantly reduces cell death in cortical neurons of the R6 / 1 huntingtonian model (Fig. 7 ). In addition, as seen in Figure 3, there is a massive Ca ^{++ entry} in a large number of terminals, a situation that is repeated in cortical neurons transfected with the N-terminal region of the exon-1 of the HT expressing 72Q but which can be completely blocked by the BBG. The massive entry of Ca ⁺⁺ produces excito-toxicity by activation of caspases, mitochondrial damage and cell death.

On the other hand, in the present invention 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.

Finally, in the present invention 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).

Therefore, 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). In addition, 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).

Thus, 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. At the mRNA level, the proportion of P2X7 messenger RNA with respect to an internal control (mRNA of β-actin) is determined by the RT-PCR technique or others that serve the same purpose. At the protein level, the proportion of P2X7 protein with respect to an internal control, such as the concentration of beta-actin, alpha-tubulin or GAPDH protein, 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

For any of these determinations the result of increased concentrations of P2X7 would result from the proportion with respect to an internal marker / control of proteins or standard mRNA, the expression of which is not modified in the course of the disease. Therefore, samples of individuals controlling the concentration of the messenger or the P2X7 protein would not be strictly necessary, but it is not ruled out that such controls could facilitate the determination. Increases in mRNA or protein in which p <0.05 according to a comparative ANOVA test were considered statistically significant. 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. , wherein 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).

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).

In the methods described above, 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.

As mentioned above, an increase in the transcription and concentrations of P2X7 in certain brain areas of murine models affected by HD is evidenced in the present invention. Thus, 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. Thus, that ligand or probe is capable of being bound to the antagonists or inhibitors of the P2X7 receptor, described in the present invention (for example BBG), or to drugs comprising said antagonists that selectively bind P2X7 and use them to visualize the concentrations of P2X7 in vivo. Therefore, 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.

DESCRIPTION OF THE FIGURES

Figure 1

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- α tubulin (α-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.

Figure 2

In the present figure, the location of P2X7 in cortico-striatal projections can be observed.

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-βlll 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).

F. 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 μm; C and D 25 μm.

Figure 3

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).

D. The quantification of the synaptic terminals that respond to the stimulation with BzATP of control animals (white bar) and Tet / HD94 (black bar) is represented.

E and F. The means of fluorescence (Fura-2) recorded over time of the two different response kinetics of the terminals that respond to stimulation with BzATP are shown. The upper bars indicate the periods of stimulation analyzed with 10μM of BzATP. The percentages of the two types of response found in the different populations of synaptic terminals are also indicated. All the terminals analyzed responded to stimulation with 3OmM K + (used as a control of the terminal's functionality). * p <0.01. (s / div: seconds / scale division).

Figure 4

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 μM 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 0.5μM and 10 µM 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 μM BzATP in cortical neurons was suppressed when the neurons were previously incubated with 1 μM 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.

(s / div: Seconds / scale division)

Figure 5

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 μM 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 µM 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. Figure 6

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.

A. BBG concentrations were measured in both blood (left) and brain (right) of treated animals, with levels of 2.25μm 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.

Figure 7

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.

A-D Immunohistochemical detection of apoptotic neurons labeled with anti-caspase-3.

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.

EXAMPLES

EXAMPLE 1. Increased levels of P2X7 in the striatum and blood of mouse models of Huntington's disease.

Immunoblot analysis of brain and striatal cortex extracts of two different mouse models of Huntington's disease (conditional model Tet / HD94 and R6 / 1), and control animals with antibodies against P2X7 (Figure 1) was performed. The membranes were incubated with anti- α tubulin to correct any possible deviation in the amount of protein. Histograms show densitometric 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 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.

Cortical and striatal sections of 16-month-old Tet / HD94 mice were performed which were labeled with anti-P2X7 antibody (Figure 2). Triple immuno fluorescence was performed with anti-P2X7 and anti-βlll tubulin antibodies and Dapi nuclear marking of cortical sections of 16-month-old Tet / HD94 mice, showing apoptotic nuclei of pyramidal neurons expressing P2X7 and signaling cortical projections expressing P2X7. A 10-month-old R6 / 1 mouse brain sagittal section was performed showing P2X7 dizziness at the cortical level, at the level of the corpus callosum and at the striatum. Finally, a double immunofluorescence was performed with anti-P2X7 and anti-DARPP-32 at the level of the R6 / 1 mouse striatum of 10 months of age.

EXAMPLE 3. Increased expression and altered response of P2X7 in synaptic terminals of the anterior brain of Tet / HD94 mice.

A double immunofluorescence of synaptic anterior brain terminals marked with anti-synaptophysin and anti-P2X7 of control animals and Tet / HD94 was performed (Figure 3). Quantification of positive synaptic terminals for P2X7 was performed in control mice and Tet / HD94 mice. On the other hand, the synaptic terminals that respond to BzATP stimulation of control mice and Tet / HD94 mice were quantified. Subsequently, the means of the fluorescence recorded over time, of the two different response kinetics of the terminals that respond to the stimulation with BzATP were measured, indicating the stimulation periods analyzed with 10 μM of BzATP and the percentages of the two types of response found in the different populations of synaptic terminals. All the terminals analyzed responded to stimulation with 3OmM K + (used as a control of the terminal's functionality). * p <0.01. EXAMPLE 4. BzATP treatment increases cell death in cortical neurons 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 μM 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 µM 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.

EXAMPLE 5. In vivo treatment of huntingtonian mice R6 / 1 with BBG.

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 2.25μm 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.

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.

Claims

1. Diagnostic and / or prognosis method, in vitro, of Huntington's chorea, characterized in that it enables the detection of a statistically significant increase (p <0.05) in the expression and / or activity of the P2X7 gene at the neuronal level , detectable in samples of cerebrospinal fluid or peripheral blood by:

a) measurement of the ratio of P2X7 mRNA to an internal control; or b) measurement of the P2X7 protein concentration with respect to an internal control; c) comparison of these values with standard values obtained from normal individuals.

2. In vitro diagnostic and / or prognostic method according to claim 1, wherein the measurement of the mRNA ratio is performed by the RT-PCR technique.

3. In vitro diagnostic and / or prognostic method according to claim 1, wherein the measurement of the protein concentration is carried out by Western-type transfer, luminometric or fluorometric techniques, or flow cytometry.

4. 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.

5. Use of P2X7 receptor antagonists capable of crossing the blood brain barrier, and pharmaceutically acceptable vehicles, for the preparation of drugs intended for the treatment of Huntington's chorea.

6. Use according to claim 5, wherein said antagonist is Brilliant Blue-G (BBG).

7. Use according to claim 5, wherein said antagonist is KN-62.

8. Use according to claim 5, wherein said antagonist is decavanadate.

9. Use according to claims 5-8, wherein the antagonist is presented bound to a molecule visible in the brain by neuro imaging techniques.

10. Use according to claims 5-9, wherein said medicaments are designed to be administered by a route other than direct administration to the central nervous system.

11. Use according to claim 10, wherein said medicaments are designed to be administered intravenously.

12. Use according to claim 10, wherein said medicaments are designed to be administered intraperitoneally.

13. Use according to claim 12, wherein the antagonist administered is BBG at a dose of 45.5 mg / kg body weight.

14. Pharmaceutical composition comprising a P2X7 receptor antagonist capable of crossing the blood brain barrier, and a pharmaceutically acceptable carrier, intended for the treatment of Huntington's chorea.

15. Pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable carrier is suitable for administration of the composition by a route other than direct administration to the central nervous system.

16. Method of in vitro detection of drugs useful for the treatment of Huntington's chorea, which decrease neuronal alterations induced by P2X7 activating substances, characterized in that it comprises the following stages: a) measure the increase in calcium concentration in the neuronal 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 the values in the presence and absence of the drug; c) consider those drugs in which said difference is statistically significant (p <0.05).

17. Method according to claim 16, wherein the proportion of neuronal apoptosis is detected by staining with propidium iodide and calcein

18. Method of identifying effective doses of P2X7 inhibitors for the treatment of Huntington's chorea, which comprises the following stages: a) measuring the concentration of internal calcium and / or the proportion of apoptosis in a culture of neurons expressing huntingtin mutated, using these measures as a control; b) in parallel, administer a dose of the inhibitor, to a parallel culture of neurons such as that of stage a); c) measure the calcium concentration and / or the proportion of neuronal apoptosis in the culture of stage b); d) identify the doses that produce a calcium concentration and / or a significantly lower apoptosis ratio (p <0.05) than those detected in stage a).

19. A method according to claim 18, wherein the proportion of neuronal apoptosis is detected by staining with propidium iodide and calcein.

20. Neuroimaging probe that allows to detect the increase in the concentration of P2X7 in the patient's brain, which comprises a ligand that selectively binds to the P2X7 receptor and is linked to a molecule that can be visualized by neuroimaging techniques.

21. Probe according to claim 20, wherein the ligand is an agonist or antagonist of the P2X7 receptor that crosses the blood brain barrier.

22. Probe according to claim 21 wherein the ligand is the P2X7 antagonist Brilliant Blue G (BBG).

23 Probe according to claim 21, wherein the ligand is the P2X7 KN-62 antagonist.

Probe according to claim 21, wherein the ligand is the P2X7 decavanadate antagonist.

25. Method of obtaining neuro images of a human being in vivo, using the probe described in claims 20-24, wherein the probe allows to detect the increase in P2X7 in individuals suffering from Huntington's chorea.