RU2614697C1 - Neuroprotective agent - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: using the lithium salt of ascorbic acid is proposed to protect neurons from glutamate stress.

EFFECT: use of the invention allows to increase the effectiveness of the neurons protection from glutamate stress with reduced toxicity.

1 tbl, 8 dwg, 2 ex

Description

The invention relates to the field of pharmacology and can be used for the prevention and treatment of diseases associated with the occurrence of glutamate stress.

Glutamate stress is not an analogue of the psychological and maladaptive concept of “stress distress” formulated by G. Selye. (Selye G. Stress without distress, M .: Progress, 1979, 123 pp. Glutamate stress is the universal response of central and peripheral nervous system cells to the effects of excessive (non-physiological) concentrations of excitatory amino acids - glutamate and aspartate - due to ischemic (impaired adequate blood supply brain region, stroke focus), genetically determined immunoneurotoxic damage (multiple sclerosis, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Alzheimer's disease, glauco optic neuropathy), temperature, physical (bruised, cut, lacerated, gunshot wound, rupture, compression of brain tissue, etc.) Glutamate stress helps accelerate the death of neurons and is one of the risk factors for neurodegeneration, reduces cell survival in adverse conditions, increases the area of brain damage, interferes with brain regeneration processes .. Glutamate stress leads to the development of a cascade of excitotoxic damage leading to the death of nerve cells. Under the influence of high concentrations of glutamate, hyperactivation of NMDA and AMPA receptors is observed on the surface of damaged cells, then an excess amount of calcium ions enters the cell, which activates a number of enzymes (proteases, endonucleases and phospholipases), the cytosolic structures of the cell collapse, and, as a result, the cell starts the process of self-destruction (apoptosis). Glutamate is the main excitotoxin with a clear dose dependence: the higher the concentration of glutamate, the larger the focus and the harder (including irreversibly) damaged neurons.

It is known the use of lithium salts in the composition of drugs for the treatment of neurodegenerative diseases, where it is used as an auxiliary component (US 20070298129, US 20070049565, US 20050233010).

Lithium chloride is known to have a neuroprotective effect on glutamate toxicity of cerebellar neurons (Shao L., Cui J., Young LT and Wang JF, The effect of Mood stabilizer lithium on expression and activity of glutathione-S-transferase isoenzymes, Neuroscience. 2008. v. 151 (2), p. 518-524).

The presence of lithium cations cardio and nephroprotective properties. The therapeutic mechanisms of the action of lithium salts in conditions associated with circulatory arrest are studied, which opens up the possibility of using lithium salts in anesthesiology and resuscitation (V.V. Moroz et al. Cell damage and protection mechanisms for ischemia / reperfusion and experimental justification for the use of lithium-based drugs in Anesthesiology, Review, General Reanimatology, 2013, IX. 1 pp. 63-72).

The existence of neuroprotective and neurotrophic synergies between lithium cations and neuropeptides of cerebrolysin is known. It has been experimentally confirmed that the neuropeptides of cerebrolysin potentiate the targeted delivery of lithium ions to the brain and their uptake by neurons (OA Gromova et al. Pharmacokinetic and pharmacodynamic synergies between neuropeptides and lithium in the implementation of the neurotrophic and neuroprotective effects of cerebrolysin. Neurology and Psychiatry 2015, No. 3, 2015) 3. .

However, the well-known publications do not trace the influence of the anion with which the lithium cation is associated on the neuroprotective properties of salts.

The closest in technical essence and the achieved result to the proposed invention is a technical solution in which it is proposed to use lithium salt of comenic acid as a neuroprotective agent and means of protection against glutamate stress of neurons. Information is presented on the effect of lithium salt of comenic acid on glutamate cytotoxicity in the culture of granular cells of the cerebellum of rat pups. In this case, the effect of coment lithium on glutamate cytotoxicity in the culture of neurons of the cerebellum of rat pups was evaluated in comparison with the actions of comenic acid and lithium chloride separately. The authors indicate a greater effect with the use of coment lithium compared with comenic acid and lithium chloride (RU 247722, 2013).

A disadvantage of the known agent is that the anion of comenic acid is not an endogenous molecule of the metabolome of the human body. Due to this, the anion of comenic acid needs biotransformation and removal of degradation products of the used agent from the body.

In addition, an increase in the number of patients with various neurological pathologies in recent years (for example, cerebrovascular pathology, ischemic stroke, traumatic brain injury, neurodegenerative diseases), in which there is an increase in the release of glutamate and aspartate by neurons, requires an expansion of the arsenal of available drugs with pronounced properties for protecting neurons from elevated levels of glutamate (the so-called glutamate stress of neurons).

The objective of the present invention is to provide an affordable neuroprotective agent with an increased degree of protection of neurons from glutamate stress with reduced toxicity.

The problem is solved by the described means of protecting neurons from glutamate stress, which is used as a lithium salt of ascorbic acid.

Information on the use of the lithium salt of ascorbic acid (lithium ascorbate) to protect neurons from glutamate stress was not found by the authors in the prior art.

It was previously proposed to use lithium ascorbate in the following areas of medicine. It is known to use lithium ascorbate as a hemaprotective agent (RU 2351326, 2009), as a means of increasing the activity of neutrophils (RU 2226391. 2004). Lithium ascorbate is also recommended for use as part of agents with antibacterial, antioxidant, and immunostimulating activity (RU 2535140, 2014, RU 2372081, 2009, RU 2444358, 2012).

The lithium salt of ascorbic acid used by the authors was obtained as follows.

A suspension of ascorbic acid in distilled water is prepared. With heating and constant stirring, lithium carbonate, or lithium hydroxide, or both of the above lithium compounds are portioned into the suspension. The suspension is heated to completely dissolve the components of the reaction mixture, crystallization of the precipitate, washing and drying are carried out. The dried salt crystals are subjected to double recrystallization in a solution of ethyl or isopropyl alcohol under cooling, followed by washing with alcohol and drying. As a result, high-purity lithium ascorbate was obtained with a yield of up to 95%. The use of the double recrystallization method of salt in the indicated alcohol solutions in the process of obtaining lithium ascorbate made it possible to obtain a product that does not contain extraneous anions in the form of hydroxide or carbonate, which contributes to an additional effect regarding the reduced toxicity of the obtained agent. The high yield of the target product allows you to scale the technology of salt production to industrial production.

The results of in vivo experiments are presented below as examples, confirming the presence of high adaptogenic properties in lithium ascorbate, as well as the results of the effect of lithium ascorbate on the survival of neurons in vitro under conditions of glutamate stress.

The invention is further illustrated by specific examples and illustrated in the figures, which represent:

In FIG. 1 - Fixed cerebellum cell culture stained with trypan blue.

In FIG. 2 - Dose-dependent glutamate protective effect of lithium ascorbate.

In FIG. 3 - Empirical distribution functions of the number of survivors of SCW at various concentrations of lithium ascorbate (mm).

In FIG. 4 - Neuroprotective effect of lithium ascorbate during the cytotoxic effect of glutamate (100 mM) on SCN.

In FIG. 5 - Neuroprotective effect of lithium ascorbate during the cytotoxic effect of glutamate.

Figure 6 - Neuroprotective effect of lithium ascorbate introduced into the culture 3 hours before the cytotoxic effect of glutamate.

In FIG. 7 - The number of surviving neurons, calculated on stained preparations.

In FIG. 8 - Hyperphosphorylation of tau protein in the hippocampus of rats under chronic stress compared with control.

Example 1

The experiment used 7-8-day-old cultures of granular cerebellar neurons obtained by enzyme-mechanical dissociation of cerebellar cells of 7-day-old rats according to the method [Andreeva N.A., Stel'mashuk E.V., Isaev N.K., Ostrovskaya R.U., Gudasheva T.A., Viktorov I.V. Neiroprotektornye effekty nootropnogo dipeptida GVS-111 pri kislorodno-glyukoznoi deprivatsii, glutamatnoi toksichnosti i oksidativnom stresse in vitro. // Byull. eksperim. biol. med. 2000.V. 130. No. 10. from. 418-421.]. The rats were subjected to a lethal dose of ether anesthesia, after which they were sterilized with 70% alcohol for 5 minutes, the cerebellum was removed and transferred to a plastic Petri dish with a phosphate buffer devoid of calcium and magnesium ions. Tissue fragments were incubated for 15 min at 37 ° C in phosphate buffer containing 0.05% trypsin, 0.2% EDTA and 0.8% glucose. After incubation, the tissue was washed in two shifts of phosphate buffer and once in a culture medium, in which it was then subjected to mechanical dissociation. The composition of the medium included 90% of the minimal Igla medium, 10% fetal calf serum, 2 mM glutamine, 5 mM KCl and 10 mM Hepes buffer, pH 7.2-7.4.

The cell suspension was centrifuged for 1 min at 1000 rpm, the supernatant was removed, and the pellet was resuspended in a nutrient medium. Cultivation was carried out in 96-well plastic plates coated with polyethyleneimine or polylysine in a nutrient medium, where the level of potassium chloride was brought to 25 mm. 0.1 ml of cell suspension was added to each cell of the plate.

Cultivation was performed for 7-8 days in a CO ₂ incubator filled with a gas mixture (95% air + 5% CO ₂ ), at a temperature of 35.5 ° C and a relative humidity of 98%. By this time, cultured granular neurons (CZN) reach their morphological and neurochemical maturity. The condition of the cultures was monitored daily and at each stage of the experiment by visual viewing in an inverted microscope with phase contrast.

A lithium ascorbate solution was prepared from a freshly prepared dry substance obtained by the method described above, immediately before being introduced into the culture medium. The drug was weighed, dissolved in PBS, the solution was sterilized by filtration and added to the nutrient medium on the second day in vitro for the entire cultivation period (up to 7 days). Based on the initial concentration of the substance (10 mM), its minimum possible dilution for addition to the cultures, provided that the necessary properties of the nutrient medium were preserved, was 1:10, i.e. 1 mM. 96-well plastic plates allowed 4 different concentrations of samples to be tested immediately. The following concentrations were selected: 0.1, 0.2, 0.5, and 1.0 mM.

Grain cerebellar neurons were easily identified in cultures as small, 7-10 microns in diameter, round or oval cells in cultures. Survival was quantified using direct scoring of CZN with normal morphology on fixed trypan blue stained preparations. On these preparations, the nuclei of neurons are clearly visible, occupying a large part of the cell body and surrounded by a thin rim of the cytoplasm (see Fig. 1).

In FIG. 1 shows a fixed cerebellar cell culture stained with trypan blue. A - control, B - 24 hours treatment with glutamate. Green arrows indicate CZN with normal morphology, yellow - glial cell nuclei, red - pyknotic nuclei of dead CZN. Scale 15 microns.

For each point, 3 cultures were taken, in each of which 5 fields of view were photographed and calculated successively (at least 45 fields from 9 cultures in 3 independent experiments). The number of neurons with unchanged morphology in control cultures was taken as 100% survival. For statistical analysis, we used the ANOVA test with Bonferroni / Dannett corrections and the non-parametric Kolmogorov-Smirnov test, which is highly sensitive and is applicable to the analysis of samples of numbers regardless of the fulfillment of the normality distribution condition. Differences between groups were considered significant at p <0.05. The results were expressed as mean ± standard deviation.

Glutamate (Sigma, USA, N.G-1626), when added to cultures, had a dose-dependent toxic effect (see FIG. 2).

In FIG. Figure 2 shows the effect of glutamate (Glu) on crop survival (counting morphologically unchanged neurons on fixed preparations stained with trypan blue).

The choice of concentration (out of the three used) was carried out in each experiment in such a way that the survival rate of CZN was 30-80% of the control. It is this survival rate that makes it possible to identify the effect of various substances on the process of cell death. With a survival rate of less than 30 or more than 80%, neuroprotective effects may not be detected. The studies used cell counting under the action of putative neuroprotectors, adding selected toxic concentrations of glutamate (100-500 μM) to the nutrient medium.

Dose-dependent glutamate protective effect of lithium ascorbate

As shown by the analysis of the empirical distribution functions of the averages using the Kolmogorov-Smirnov test, lithium ascorbate (without the addition of glutamate) in the cerebellum cell culture in a concentration range from 0.1 to 1.0 mM is non-toxic to CZN (see Fig. 3).

The figure 3 presents the empirical distribution function of the number of survivors of SCW at various concentrations of concentrations from 0.1 to 1.0 mm lithium ascorbate (mm). In a cerebellum cell culture, lithium ascorbate (without glutamate addition) is not toxic to CZN.

Kolmogorov-Smirnov test

In a cerebellum cell culture, lithium ascorbate (without the addition of glutamate) in a concentration range from 0.1 to 1.0 mM is not toxic to CZN.

Using figures 4-6 illustrated the solution to the problem with the achievement of the claimed technical result

The figure 4 shows the neuroprotective effect of lithium ascorbate with the cytotoxic effect of glutamate (100 mm) on CZN. Empirical distribution functions of numbers (Kolmogorov-Smirnov test) of surviving SCN under the action of glutamate in the absence (Glu) and in the presence of (Glu +) lithium ascorbate in the concentration range from 0.1 to 1.0 mM. Significant differences are shown for all used concentrations of lithium ascorbate compared with the action of glutamate without the addition of the drug. The values of the maximum deviation “D” are indicated, on the basis of which the statistical significance of the differences between the distribution functions is calculated.

The figure 5 shows the neuroprotective effect of lithium ascorbate during the cytotoxic effect of glutamate. White bars are lithium ascorbate (0.1-1.0 mM) in the absence of glutamate, black bars at the same concentrations in the presence of 100 μM glutamate (Glu). To - control. For each column, 30 fields of view were calculated. * - p <0.01 compared with glutamate without the addition of the drug.

An analysis of the data using the ANOVA test showed that the survival of neurons during the cytotoxic effect of glutamate in the presence of lithium ascorbate in the concentration range from 0.1 to 1 mM, starting from a concentration of 0.2 mM, significantly increased on average by 12%, and a dose-dependent effect was observed ( see Fig. 5).

The neuroprotective properties of lithium ascorbate were studied when it was added to cultures just 3 hours before exposure to glutamate. In accordance with the results of the ANOVA test, a protective effect was found, although less pronounced, in which neuron survival was significantly increased by 8% at a drug concentration of 0.5 mM (Fig. 6).

Figure 6 shows the neuroprotective effect of lithium ascorbate introduced into the culture 3 hours before the cytotoxic effect of glutamate. White bars are lithium ascorbate (0.1-1.0 mM) in the absence of glutamate, black bars at the same concentrations in the presence of 100 μM glutamate (Glu). To - control. For each column, 15 fields of view were calculated. * - p <0.01 compared with glutamate without the addition of the drug.

The figure 7 shows the number of surviving neurons, calculated on stained preparations, which clearly shows that the presence in the nutrient medium of lithium ascorbate prevents the destruction of SCN under the influence of glutamate. In this figure, the neuroprotective effect of lithium ascorbate with the neurocytotoxic effect of glutamate (100 μM, 24 h) in cerebellum cell culture. KZN is indicated by arrows. Neurons after apoptosis look like circles of a dark shade with a smaller radius than the surviving neurons. Color fixed cultures trypan blue. Scale 15 microns.

Analysis of the data presented in the form of illustrations showed the following.

Under the cytotoxic effect of glutamate (100 μM), lithium ascorbate in the studied concentration range had a pronounced neuroprotective effect. The results of the analysis of the distribution functions of the average numbers of surviving neurons at all concentrations of lithium ascorbate showed a significant difference from the results obtained with the action of glutamate without adding the drug (Fig. 5). The use of even a minimal concentration of lithium ascorbate (0.1 mM) led to a significant difference (D = 0.19, P = 0.049). With an increase in the concentration of the claimed drug, the severity of differences increased (the values of the maximum deviation "D" increased and the P values decreased), and at the highest concentration (1.0 mmol) the differences between glutamate toxicity and intact control were on the verge of statistical significance (D = 0 , 17, P = 0.059).

Thus, lithium ascorbate in the entire studied range of concentrations (0.1-1.0 mm) significantly increases the survival of neurons when exposed to glutamate.

Comparative studies of lithium salts with different anions were carried out. Studies have shown the following.

The use of lithium chloride and coment lithium also showed a protective effect under glutamate stress, but neuron survival was significantly lower (p = 0.0048, p = 0.006). Lithium carbonate did not have a protective effect at all and even reduced the survival of neurons against the background of glutamate concentrations of 250 μM and 500 μM. The low bioavailability of lithium cations associated with other anions of inorganic acids does not protect cells well, especially at high concentrations of glutamate. Lithium coment taken as a prototype of the proposed invention has a pronounced dose-dependent protective effect at glutathate concentrations of 50 ... 250 μM, but compared with lithium ascobate, coment lithium is a significantly less effective means of protection against glutamate stress.

The obtained comparative results are shown below in table 1.

The dynamics of changes in the survival of neurons in percent exposed to glutamate at a concentration of 50, 100 and 250 μM against the background of the preventive introduction (3 hours before exposure to glutamate) of lithium preparations in a neurocyte culture.

Example 2

Keeping animals in a very tight cage, excluding the movement of the animal - the so-called immobilization stress - provokes the development of degeneration of nerve structures. Chronic immobilization stress for 4 weeks led to an increased release of glutamate and hyperphosphorylation of the tau protein in the hippocampus. Other signs of glutamate-induced neurodegeneration were a decrease in the levels of structural neuronal proteins — a protein associated with microtubules (map2) and gamma synuclein. The identification of hyperphosphorylated tau protein using antibodies confirmed the data of immunohistochemistry and indicated an increase in its level during chronic immobilization, accompanied by an increased release of glutamate for 4 weeks, compared with a group of animals also exposed to chronic immobilization for 4 weeks, but receiving lithium ascorbate in a dose of 1 ... 50 μg / kg of body weight is illustrated in FIG. 8. Subsidies of lithium ascorbate 1 ... 50 μg / kg body weight for 4 ... 12 weeks before the experiment and 1 ... 6 weeks after showed a pronounced protective effect.

The figure 8 presents data on the hyperphosphorylation of tau protein in the rat hippocampus under conditions of 4 weeks of chronic immobilization of stress (Fig. Above) and with the preventive use of lithium ascorbate at a dose of 30 μg / kg of weight for 4 weeks before the creation of the model (Fig. Below) .

A. Immunohistochemical identification of hyperphosphorylated tau protein (anti-PHF). Hematoxylin staining of nuclei. Scale 200 microns.

B. Identification of hyperphosphorylated tau protein using antibodies (immunoblot).

The data obtained by the authors indicate the expressed adaptogenic properties of lithium ascorbate under stress modeled in vivo and in vitro, and the close relationship between stress and neurodestructive processes at the cellular level induced by hyperactivation of glutamate receptors.

Data on the protective effect of lithium salts based on comenic acid (prototype agent) on the levels of structural neuronal proteins associated with the effects of glutamate on proteins supporting microtubule synthesis (map2) and the level of gamma synuclein are absent in the prototype.

Analysis of the above results confirms the achievement of the claimed technical result in the amount of the essential features included in the claims.

Claims (1)

Neuroprotective agent for protecting neurons from glutamate stress based on lithium salt, characterized in that the lithium salt of ascorbic acid is used.

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