The use of agonists or antagonists of p2-purinoceptors for the prevention of cytotoxicity caused by glutamate

 

The invention relates to medicine, specifically to pharmacology. Proposed: the use of agonists or antagonists of P2-purinoceptors for the prevention of cytotoxicity caused by glutamate, for modulation caused by glutamate physiological functions and pharmacological composition with these new properties. The agonists R2-purinoceptors include 5-adenosinetriphosphate, the antagonists - reagent baseliney blue and zibarro blue 3GA. For the first time revealed the inclusion of purinoceptors in the development process glutamatnami neurotoxicity. The invention expands the Arsenal of tools specified destination. 5 S. and 6 C.p. f-crystals, 10 ill.

BACKGROUND of the INVENTION the Invention relates to the use of a specific class of compounds to prevent the development of cytotoxicity caused by glutamate.

PRIOR art Glutamate is the main excitatory neurotransmitter of the Central nervous system (Hollmann M, Heinemann, S., Annu. Rev. Neursci. 11, 31-108, 1994), and the widespread distribution of glutamate receptors throughout the Central nervous system asserts the fact that glutamate plays a Central role in the mechanism of various fiziologicheskii and individual experimental data suggest a Central role-dependent glutamate neurotransmission in such bodily functions, as training, recognition and memory (Bliss T. V. P, Collingridge G. L., Nature 361, 31-39, 1993).

In addition, in the last decade were obtained evidence that glutamate is toxic to neurons in vivo and in cell culture, and that the functioning of glutamate receptor is crucial for the development of numerous diseases and lesions of the brain (Appel S. H., Trends Neurosci. 16, 3-5, 1993). Many neurological diseases, including stroke or epileptic seizures cause brain damage due to enhanced stimulation of its glutamate, and degenerative diseases, which include Alzheimer's disease, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis (ABS), accompanied by neuronal cell death caused by excessive activation of glutamate receptors.

The PURPOSE of the INVENTION the present invention is the provision of a specific class of compounds for modulation caused by glutamate neurotransmission and neurotoxicity, which would make possible the treatment of acute and chronic neurodegenerative diseases.

Another objective of the present invention is the provision of a specific class of compounds, making it possible modulation wired Morehouse, breathing, learning, pattern recognition and memory.

Another objective of the present invention is the provision of a specific class of compounds that could be used as a pharmacological tool for the prevention of cytotoxicity caused by glutamate.

Another objective of the present invention is the provision of a specific class of compounds, which would be valid pharmacological alternative to the previously described compounds, such as competitive antagonists (antimetabolites) and non-competitive antagonists of glutamate, gangliosides and growth factors for the treatment of acute and chronic neurological disorders associated with glutamate.

Description of the INVENTION These and other objectives and associated benefits, which are more clearly marked in the description below, can be achieved using compounds that are agonists or antagonists of P2-purinoceptors, for the prevention of cytotoxicity caused by glutamate.

An essential novelty of the present invention is to identify the correlation between the biological effects caused by glutamate, and modulators of Ptotal property, on the basis of which they can be attributed to both ionotropic and metabotropic types of receptors.

As an example, the selected connection baseliney blue E-3G (also called Reactive blue 2) and zibarro blue 3GA, which are antagonists of P2-purinoceptors. These compounds can be obtained, for example, from Sigma (Sigma), and their molecular structure and main characteristics of the described distributed in Italy the company directory Sigma 1995, respectively, on page 149 for basilikovogo blue E-3G and on page 266 for cibachrome blue 3GA. As another connection was selected 5-adenosinetriphosphate (AMPPNP), which is an agonist of the P2-purinoceptors. This connection can be obtained from the company Sigma, and its molecular structure and main characteristics are described on page 52 catalog Sigma, published in Italy in 1995.

In accordance with the present invention these compounds are used to prevent caused by glutamate cytotoxicity in cells of the Central nervous system, in particular in neurons of the Central nervous system. As a model cell system for neurons in the CNS was used cerebellar neurons in postnatal rats. These cells, therefore, jacka postnatal rats (R. S. Lasher, and Zagon I. S., Brain Res. 41, 428-438, 1972) with the growth in vitro to form a Mature phenotype in the form of interneurons that use glutamate as a neurotransmitter and, in addition, represent an excellent model system for studies of mediated glutamate cytotoxicity.

Impact on granular neurons glutamate in the amount of 100 microns within 15-30 minutes leads (15-20 hours) to the death of 80-100% of the cells from their total number. Found that the antagonist P2-purinoceptor baseliney blue, also known as reactive blue 2 (derived anthraquinone-sulphonic acid), at its introduction in granular neurons in the amount of 100 μm under the condition of simultaneous presence of glutamate fully supports survival of cells, thus eliminating the cytotoxic action of glutamate. Impact basilikovogo blue on the morphology of the granular cells of the cerebellum demonstrates, despite the presence of glutamate, which otherwise induces a complete cell death, very healthy cell cells, which participate in the operation of a dense network of highly branched processes. Baseliney blue also contributes to the preservation of adhesion and fasciculation of natraul. Acute reaction, the situations neurons within the first five minutes after treatment with glutamate, also can be avoided by adding basilikovogo blue, and this suggests that this compound are, apparently, at a very early stage in the chain of events, occurring immediately after exposure to EAA receptor (the receptor induced by amino acids).

It is important to emphasize that baseliney blue up to the highest investigated concentration component 300 μm, is not toxic to cells, and the introduction of it into granular neurons for a period of from 0.5 to 26 hours does not affect the permeability of the plasma membrane (which is measured by the level of absorption of ethidiumbromid) or on cellular metabolism (which is determined by the conversion of MTT (3-(4,5-Dimethylol-2-yl)-2,5-diphenyl-tetrazolyl bromide) in formazan due to mitochondrial dehydrogenase activity). Baseliney blue prevents cell death caused by glutamate, demonstrating the value IR50in the range of 10-20 μm, which is basically consistent with the concentrations of the compounds listed as an antagonist of P2-purinoceptor.

Other commercially available isomer sulfonic derivative of anthraquinone (zibarro blue) are also effective in this regard. Caffeine antagonist P2

Baseliney blue inhibits the binding of [3N]-ATP by membrane granular neurons with a value of IR50about 10 μm, which corresponds to the value of IR50that is the prevention of the development of cytotoxicity caused by glutamate. Research associate with [3N]-ATP was also carried out directly on intact cells, it was shown that baseliney blue also effective.

It was conducted by culturing cells at a constant presence from the first day, but no later than the second day in vitro) 100 μm 5-adenosinetriphosphate (AMPPNP) as a known agonist of the P2-purinoceptor. Under this treatment induced glutamate cytotoxicity is inhibited by about 50-60%. Acute exposure of cells to 100 μm AMPPNP (simultaneously with glutamate) is not effective in this respect. The fact that the cultivation of neurons in the continuous presence of AMPPNP achieves the same effect, which occurs when the acute effects basilikovogo blue, supports the hypothesis that the direct inclusion of purinoceptors in the process razvi likely is the phenomenon of desensitization of purinoceptors.

Since the release of D-[3H]-aspartate is often used to assess functional status of granular neurons of the cerebellum in vitro and the release of aspartate-induced depolarization or glutamate, is an ability, progressive purchased these cells together with the maturation of neurons, it was decided to explore this option for a more in-depth study of the biological effects and possible mechanism of action basilikovogo blue, which is used to prevent cell death. Discovered that baseliney blue inhibits glutathionine the release of [3H] -aspartate value IR50about 10 microns. Inhibition is almost complete, but it does not affect the main release, which takes place during the measurement of the release within one minute, over a longer period (3, 10 and 25 minutes) and even in the presence of MD2+. Chronic effects on granular neurons within 8 days AMPPNP in the amount of 100 μm also leads to inhibition by 70-80% glutamatinduzierter release of [3H]-aspartate.

Glutaminovaya neurotoxicity is often accompanied by an increase in granulosity blue, unlike caffeine, virtually eliminates caused by glutamate, but not the main absorption of CA2+with the value IR50approximately 10 microns. This value is also in accordance with the value IR50shown in relation to inhibition of ATP binding, cytotoxicity and release of aspartate. Dependent vazelinovogo blue inhibition occurs when measuring the absorption of Ca2+within a short (1 minute) or a longer period of time (3, 10 and 25 minutes). Chronic effects on granular neurons within 8 days 100 mm AMPPNP leads to inhibition by 50-70% glutamatinduzierter influx of Ca2+similarly, the inhibition of cytotoxicity and release of aspartate.

Description of the DRAWINGS Baseliney blue prevents the development of cytotoxicity induced by glutamate in primary cultures granular cells of the cerebellum: shows the dependence of the effect of dose and route of administration. Can replicate culture granular cells of the cerebellum when breeding 8 DIV were exposed to the effect for 25 minutes 100 μm glutamate at simultaneous presence of different concentrations basilikovogo blue (Fig.1). After 20 hours was evaluated vaivaikathashtami while adding to the cells 100 μm 100 μm glutamate and caffeine. In Fig.2 can replicate culture granular cells of the cerebellum at 8 DIV were exposed for 25 minutes 100 μm glutamate. In different periods of time after selection of glutamate to the medium was added baseliney blue (100 μm) and after 20 hours in cell cultures was assessed by the degree of survival of cells by directly counting the number of viable intact nuclei. An asterisk indicates the percentage of nuclei obtained by adding to the cells 100 μm glutamate and 100 μm basilikovogo blue. In Fig.3 can replicate culture was pre-treated with 100 μm basilikovogo blue for various periods of time before adding 25 minutes to 100 μm glutamate (carried out in the absence of basilikovogo blue). After 20 hours in the crops evaluated the survival of cells.

An asterisk indicates the percentage of nuclei observed while adding to the cells 100 μm glutamate and 100 μm basilikovogo blue. The results are presented as the average values ofThe standard deviation (n=4), and 100% of viable cells corresponds to the total number of 1.75-2106cells.

METHODS. After about 20 hours after exposure granular cells with glutamate - bromide of ammonium, 0,28% - acetic acid, a 0.5% Triton X-100, 3 mm NaCl, 2 mm MgCl2in the FBI with pH 7.4, diluted 1/10).

After 1-2 minutes the cells were washed for some time, providing a homogeneous suspension of intact viable nuclei. The last number was determined by counting in hemocytometer. Destroyed or damaged kernels in the results of the count were not included.

Baseliney blue inhibits the binding of ATP by membrane granular cells of the cerebellum, but not the uptake of aspartate. Membranes were obtained from granular cells of the cerebellum when breeding 8 DIV, and incubated 20 µg of protein with [3N] -ATP (0.5 mccoury/ml, the final concentration of 14 nm) (Fig.4) in the presence of different concentrations basilikovogo blue for one hour at 4oC. specific binding, and the values of the calculation are equal to the average value ofThe standard deviation (n=3). The asterisk denotes a linking carried out in the presence of 100 μm of caffeine. In Fig.5 can replicate culture granular cells of the cerebellum at 8 DIV washed twice and incubated for various periods of time in solution Locke with [3N]-D-2,3-aspartic acid (1 mccoury/ml, the final concentration of 40 nm and determined the amount of incorporated radioactivity by liquid scintillation counting. Presented values are given as the number of pulses per minute at 1 (ICG pulse/min/g)(cpm/g) and represent average values ofThe standard deviation (n=3). The concentration of protein was determined by the method of Bradford using standard sheep serum albumin.

METHODS. Can replicate granular cells of the cerebellum when 8DIV was collected in chilled in ice in buffer A (50 mm Tris with 1 mm EDTA, brought to pH 7.4 with Hcl containing 2 mm phenyl-methylsulfonylamino, 200 KIE/ml Aprotinin and 1 μg/ml leupeptin) and was centrifuged at 35000 x g for 20 min at 4oC. the Precipitate resuspendable in buffer a to obtain the protein concentration in the range of 5-6 mg/ml and immediately used to study the binding process. After binding with [3N]-ATP samples (1 ml) was filtered under vacuum through glass fiber filters (Whatman GF/B (Whatraan GF/B) and then immediately washed filters (34) five milliliters of 50 mm Tris-Hcl buffer (pH 7.4), dried in air and in liquid scintillation counter was assessed level of specific binding of radioactivity.

Cultivation of granular cells of the cerebellum in the presence of AMPPNP: vickye culture granular cells of the cerebellum and, since 1 DIV, some of them daily was added to 100 mm AMPPNP.

(Fig. 6): at 8 DIV after two washes some of the cultures were incubated at 20oWith over 25 min with 100 μm glutamate. The next day there was an assessment of the level of survival of the cells, as described earlier.

(Fig. 7): can replicate culture granular cells of the cerebellum at 8 DIV were incubated for one minute in a solution of Locke in the presence of 45 CA++(1 mccoury/ml) in the presence or absence of 100 μm glutamate, and then estimated the flux of CA++.

(Fig. 8): can replicate culture granular cells of the cerebellum at 8 DIV were incubated for 5 minutes in a solution of Locke in the presence of [3N]-D-2,3-aspartic acid (1 mccoury/ml, final concentration 40 nm) and then assessed the level of release of aspartate in the presence or absence of 100 μm glutamate. The data represent average values ofThe standard deviation (n=3).

Baseliney blue inhibits the release of aspartate and absorption of Ca++induced by glutamate in granular cells of the cerebellum.

(Fig. 9): can replicate culture granular cells of the cerebellum at 8 DIV were incubated for 5 minutes in a solution of Locke in the presence of whom were incubated for one minute in a solution of Locke in the presence or absence of 100 μm glutamate and in the presence of different concentrations basilikovogo blue. Buffer, removed from the culture during the phase of release, were collected in vials and determined the level of radioactivity. An asterisk indicates the value of the release of aspartate, obtained by the simultaneous presence of 100 μm 100 μm glutamate and caffeine. The cells were dissolved in 0.1 M NaOH and determined the protein concentration by the method of Bradford using as a standard of bovine serum albumin.

(Fig.10): can replicate culture granular cells of the cerebellum at 8 DIV were incubated for one minute in a solution of Locke in the presence of 45 CA++(1 mccoury/ml) in the presence or absence of 100 μm glutamate and with the simultaneous presence of different concentrations basilikovogo blue. After two washes chilled in ice 154 mm choline-chloride, 2 mm EDTA, the cells were literally in 0.1 M NaOH and collected aliquots to determine the flow of CA++(by counting radioactivity) and determine the concentration of protein. The asterisk indicates the flux of Ca++obtained in the case of the simultaneous presence of 100 μm 100 μm glutamate and caffeine. Data represent average values ofThe standard deviation (n=3).

Claims

1. The use of compounds of the W compounds are agonists or antagonists of P2-purinoceptors as an active agent for the prevention of cytotoxicity caused by glutamate.

3. Pharmaceutical composition for the prevention of neurotoxicity induced by glutamate, and modulation caused by glutamate physiological functions, including connection agonists R2-purinoceptors as an active agent in combination with the usual pharmaceutically acceptable additives.

4. Pharmacological composition according to p. 3, characterized in that it comprises connection agonists R2-purinoceptors as an active agent in addition to the commonly used pharmaceutically acceptable additives, in a pharmacologically acceptable medium.

5. Pharmacological composition according to p. 3, characterized in that these agonists R2-purinoceptors represent a 5-adenosinetriphosphate (AMPPNP).

6. The use of compounds of agonists or antagonists of P2-purinoceptors as an active agent for the prevention of neurotoxicity induced by glutamate.

7. Application under item 6, characterized in that these antagonists are selected from basilikovogo blue E-3G (Reactive blue 2) and cibachrome blue 3GA.

8. Pharmacological composition according to the

9. The use of compounds of the agonist or antagonist P2-purinoceptors as a means to modulate physiological functions caused by glutamate.

10. Application under item 9, characterized in that when the compound is an agonist of the P2-purinoceptors, these physiological function caused by glutamate, are a pain.

11. Application under item 9, characterized in that when the compound is an agonist of the P2-purinoceptors, these physiological function caused by glutamate, are a pain, hormonal balance, blood pressure, thermoregulation, respiration, exposure, recognition and memory.

 

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