RU2383615C1 - Method of screening pharmacological compounds according to neuroprotective activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: primary neuron cultures or primary glial cells are cultured. The compound under test is added. Survival rate of the cultured neurons or proliferation of the cultured glial cells is evaluated.

EFFECT: invention increases rate of screening compounds, enables search for new compounds with the highest activity which are potential neuroprotectors.

6 cl, 4 dwg, 1 tbl, 4 ex

Description

Application area

The invention relates to biotechnology. The invention allows to speed up the screening of compounds, as well as to search for new compounds with the highest neuroprotective activity.

Current level of research and knowledge

The creation of new effective and safe pharmacological agents to increase the effectiveness of mental labor in difficult conditions and to correct pathologies of the central nervous system (CNS), including neurodegenerative diseases, is especially relevant in the modern era, which is characterized by an increase in the influence of stressful factors on the human body, as well as environmental degradation in many areas of the globe. Long-term mental stress, coupled with the above factors, constantly accompanies many modern areas of human activity despite the continuous improvement of their technical support and leads to damage and death of nerve cells. The development of neurodegenerative processes in the central nervous system is also associated with various pathologies, a hereditary predisposition and traumatic brain injuries. The proportion of neurodegenerative diseases in the overall morbidity structure increases with increasing life expectancy. The most common are Alzheimer's disease and strokes. Strokes, in our opinion, can be attributed to neurodegenerative diseases in connection with the mass death of cells, in particular neurons, in the central nervous system. Currently, the number of people in the world suffering from Alzheimer's disease, according to various estimates, up to 10 million people. According to forecasts, if scientists do not find a cure for this disease, after 25 years, 22 million people on our planet will suffer from this ailment, and after 50 years - about 45 million. A similar situation with strokes. About 6 million people suffer a stroke annually, 4.7 million die. In Russia, up to 450 thousand strokes are registered per year, most of them related to severe and moderate severity. Only about 20% of surviving patients can return to their previous job.

Neurotrophins are known - a family of regulatory proteins of nervous tissue that are synthesized by neurons and neuroglia cells and contribute to the differentiation and maintenance of the viability and functioning of peripheral and central neurons. In this case, autocrine and paracrine regulatory mechanisms are used (Yuen et al., 1996). During embryonic development, neurotrophins contribute to the survival of peripheral neurons before and during the innervation of target cells. They function in the same way as physiological survival signals for central neurons. With the development of the nervous system with the help of neurotrophins, the survival of neurons is regulated, that is, in the mature nervous system there are those neurons that in the process of development of the nervous system received neurotrophins in the proper amount from the corresponding target cells. The "extra" neurons are removed by the mechanism of programmed cell death. Neurotrophins regulate neuronal differentiation, induce branching of dendrites (arborization) and axon growth (sprouting) in the direction of target cells. In the mature nervous system, neurotrophins regulate both short-term synaptic transmission and long-term potentiation (LTP), thus participating in ensuring the plasticity of the nervous system necessary for its normal functioning.

Known neurotrophin - nerve growth factor (NGF). The main targets for NGF, the first neurotrophin detected, are forebrain cholinergic neurons, which play a significant role in the central nervous system functions such as attention, learning, memory; striatal cholinergic neurons involved in movement control; sensitive neurons and most neurons of the sympathetic nervous system.

Known neurotrophin is a brain neurotrophic factor (BDNF). Identified targets for BDNF are dopaminergic neurons of the substantia nigra, cholinergic neurons of the forebrain, serotonergic neurons of the cortex, GABAergic neurons of the striatum, granular neurons of the cerebellum; motor neurons, neurons of the ciliary ganglion; neurons of the spinal nodes.

Known heptapeptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro), synthesized at the Institute of the applicant. The effectiveness of using Semax as a neuroprotective agent in the acute period of ischemic stroke has been established (Gusev et al., 1997). Semax has a pronounced neuroprotective effect, the main components of which are inhibition of glial inflammation reactions, improvement of trophic support of the brain, inhibition of nitric oxide synthesis and other oxidative stress reactions, as well as immunomodulation. The chains of metabolic transformations induced by Semax mutually reinforce and support each other, inhibiting most of the important mechanisms of delayed cell death (Skvortsova et al., 1999).

Comparison of clinical dynamics in the group with and without Semax showed a significant improvement in recovery processes during treatment with Semax in patients with moderate to severe stroke. With an ischemic stroke of moderate severity with a predominance of focal neurological symptoms, the effectiveness of Semax manifested itself from the first day of the disease and was expressed in the general activation of patients and accelerated regression of focal symptoms. In 88% of patients treated with Semax, the course of the stroke was regredient with a stable regression of neurological disorders, which significantly exceeded the same indicator in the control group (55.2%). Under the influence of Semax, by the end of the acute period of stroke in 84% of patients, a good restoration of functions was observed, and in 32% of patients the regression of focal symptoms was complete (Gusev et al., 1999). In severe stroke, the use of Semax in the first hours and days of the disease significantly relieved the regression of not only focal, but also cerebral and autonomic symptoms. In all patients who arrived in a state of stunning, a clear consciousness returned by the end of the second day. By the fifth day of stroke in 80% of patients, the disorder of consciousness and meningeal symptoms completely disappeared. In 80% of patients receiving Semax, the regredient course of the disease was determined against 62% in the control. In the Semax group, mortality in severe stroke was 10%, while in the control group it was 28.4%.

Primary cultures of eukaryotic cells are known that are used to screen compounds with various properties [1].

The task of effective search and rapid screening of compounds with neuroprotective activity is currently extremely urgent. The urgent task of identifying compounds with undesirable effects, especially promoting the proliferation of glial cells and their malignant degeneration.

Disclosure of invention

The use of primary cultures of neuronal cells allows us to evaluate the cytotoxic and neuroprotective effects of the tested compounds.

The use of primary cultures of glial cells allows us to evaluate not only the cytotoxic and neuroprotective effects of the tested compounds, but also their effect on the proliferative activity of these cells.

An additional analysis of the expression of neurotrophin genes in glial cells under the action of the tested compounds provides additional information regarding their neuroprotective properties. The use of the proposed test system allows for accelerated screening (of the order of two weeks) of a large number of compounds for the presence of neuroprotective activity in these compounds, as well as the detection of side activities of these compounds (effect on proliferation).

The implementation of the invention

The technical result achieved by the implementation of the present invention is the acceleration of testing compounds with neuroprotective activity, the identification of new compounds with neuroprotective activity, the assessment of side effects of the tested compounds at the cellular level. The specified technical result is achieved through the use of primary neuronal and glial cell cultures from the mammalian brain. This application describes the use of nerve and glial cells derived from rat brain. In addition, this technical result is achieved by evaluating the ability of the tested pharmacological compounds to stimulate the expression of neurotrophins by glial cells.

Definition of Terms

Primary neuronal culture is understood as a cell culture isolated from 16-17-day-old embryos (E16-17) of rats by a standard method [2].

Primary glial culture refers to a cell culture isolated from the brain of rats 1-3 days old by a standard technique [3].

Neuroprotection refers to the ability of a pharmacological compound to prevent the death of nerve cells.

Proliferation refers to the ability of a pharmacological compound to stimulate cell division.

Examples of own experiments

Our own studies have found that the heptapeptide of the Met-Glu-His-Phe-Pro-Gly-Pro sequence (Semax) has neuroprotective properties (RF patents No. 2124365, No. 2251429) and is used in the treatment of strokes, a disease associated with the mass death of neurons in human brain.

Own studies have shown that neurons obtained from the mammalian embryonic brain under in vitro cultivation undergo death, mainly along the path of apoptosis. The use of the Semax peptide significantly slowed down this process.

The following are examples illustrating the invention.

Example 1

We studied the ability of the Semax peptide and its natural structural analogue of ACTH (4-7) (Met-Glu-His-Phe) to slow down the process of death of neurons obtained from rat brain in vitro.

Primary dissociated cultures of rat forebrain basal nucleus neurons were obtained from 16-17-day-old embryos (E16-17) by a standard method [2]. Sprague-Dowly rats weighing 200-300 g were planted in the ratio of 1 male to 3 females after 18 pm; the day after planting was considered the first day of pregnancy. Rats were killed by carbon dioxide asphyxiation (15 min) and placed for 1 min in an 80% solution of ethanol in water. Further, all operations were carried out under aseptic conditions at a temperature of 4-7 ° C. The brain isolated from the embryo was placed in a Hanks solution and then the basal nuclei of the forebrain were isolated, freeing the tissue from the membranes. The isolated tissue was washed once with Hanks' solution and transferred to MEM / F12 (1: 1) medium containing 20% fetal bovine serum and 2 mM L-glutamine. Tissue was mechanically dissociated into individual cells by pipetting. The resulting cell suspension was washed twice with medium by centrifugation (200 g). Cells were counted in a hemocytometer (Goryaev’s chamber) and seeded with a density of 100 thousand cells / cm ² onto 12-well (or 24-well) plates treated with poly-L-lysine in 1: 1 MEM / F12 medium containing 6 g / l D-glucose, 2 mM L-glutamine, 25 mg / l insulin, 100 mg / l transferrin, 20 nM progesterone, 100 nM putrescine and 30 nM sodium selenite. The cells were cultured in a CO ₂ incubator at 37 ° C in an atmosphere containing 5% CO ₂ and 95% air. Primary neuronal cultures were similarly obtained from other parts of the brain of rat embryos (hippocampus, cortex, cerebellum, midbrain).

The primary cultures of neurons were cultured in 12-well plates in 6 parallels for each variant. The corresponding compounds, including Semax, were added in a volume of 100 μl immediately after the cells were attached to the substrate, once. Survival of cultured neurons after 7 days of cultivation was assessed by counting cytochemically or immunocytochemically stained cells in 18 random fields of each well. Cholinergic neurons were visualized by acetylcholinesterase staining. The cultures were fixed with 4% paraformaldehyde for 1 h at room temperature, washed with PBS and incubated for 48 h in 50 mM acetate buffer (pH 5.0) containing 4 mM acetylcholine iodide, 2 mM CuSO ₄ , 10 mM glycine and 0.2 mM ethopropazine . Then, the incubation medium was taken, the cells were washed with water, and a 1.25% Na ₂ S solution was added for 1-2 min. The medium was taken and a 1% AgNO ₃ solution was added for 1-2 min. The medium was taken and the plates were washed with water. The data obtained are presented in the table.

Cultivation of primary cultures of neurons. added connections control (no additives) semax (0.1 μM) semax (10.0 μM) ACTH (4-7) (10.0 μM) brain neurotrophic factor (20 ng / ml) number of neurons 110 ± 16 165 ± 20 176 ± 21 108 ± 15 183 ± 23 the number of neurons (in%) one hundred 150 160 one hundred 166

The results indicate a significant neuroprotective effect of Semax (1.5-1.6 times) compared with the control and ACTH fragment (4-7). It is important to note that the effect of the peptide was comparable to the effect of the neurotrophic factor of the brain, a well-known compound that can prevent nerve cell death. The results obtained indicate the possibility of using a primary neuronal culture for testing pharmacological compounds with neuroprotective activity.

Example 2

We studied the ability of the Semax peptide to slow down the process of death of dopaminergic neurons in primary neuronal cultures obtained from rat brain. Primary dissociated cultures of rat midbrain neurons were obtained from 15-16-day-old embryos (E15-16) by a standard method [2]. Sprague-Dowly rats weighing 200-300 g were planted in the ratio of 1 male to 3 females after 18 pm; the day after planting was considered the first day of pregnancy. Rats were killed by carbon dioxide asphyxiation (15 min) and placed for 1 min in an 80% solution of ethanol in water. Further, all operations were carried out under aseptic conditions at a temperature of 4-7 ° C. The brain isolated from the embryo was placed in a Hanks solution and then the basal nuclei of the forebrain were isolated, freeing the tissue from the membranes. The isolated tissue was washed once with Hanks' solution and transferred to MEM / F12 (1: 1) medium containing 20% fetal bovine serum and 2 mM L-glutamine. Tissue was mechanically dissociated into individual cells by pipetting. The resulting cell suspension was washed twice with medium by centrifugation. Cells were counted in a hemocytometer (Goryaev’s chamber) and seeded with a density of 100 thousand cells / cm ² onto 12-well (or 24-well) plates treated with poly-L-lysine in 1: 1 MEM / F12 medium containing 6 g / l D-glucose, 2 mM L-glutamine, 25 mg / l insulin, 100 mg / l transferrin, 20 nM progesterone, 100 nM putrescine and 30 nM sodium selenite. The cells were cultured in a CO ₂ incubator at 37 ° C in an atmosphere containing 5% CO ₂ and 95% air.

The primary cultures of neurons were cultured in 12-well plates in 6 parallels for each variant. The corresponding compounds, including Semax, were added in a volume of 100 μl immediately after the cells were attached to the substrate, once. The survival of cultured neurons after 12 days of cultivation was assessed by counting immunocytochemically stained cells in 18 random fields of each well. The results are shown in figure 1.

The results indicate a significant neuroprotective effect of Semax (4.5-5.0 times) compared with the control. The obtained results confirm the possibility of using a primary neuronal culture for testing pharmacological compounds with neuroprotective activity in vitro.

Example 3

We studied the ability of the Semax peptide to influence the proliferation of glial cells in vitro. The primary culture of glial cells was obtained according to standard methods [3]. Sprague-Dowly or Wistar rats 1-3 days old were euthanized by carbon dioxide asphyxiation (15 min) and placed for 1 min in 80% ethanol in water. Further, all operations were carried out under aseptic conditions at a temperature of 4-7 ° C. The isolated brain was placed in Hanks' solution and then the basal forebrain nuclei were isolated, freeing the tissue from the membranes. The isolated tissue was washed once with Hanks' solution and transferred to MEM / F12 (1: 1) medium containing 20% fetal bovine serum and 2 mM L-glutamine. Tissue was mechanically dissociated into individual cells by pipetting. The resulting cell suspension was washed once with medium of the same composition by centrifugation at 200 g. Cells were counted in a hemacytometer (Goryaev camera) and plated at a density of 200 tys.kletok / ^cm2 onto poly-L-lysine culture flasks of 75 ^cm2. The cells were cultured in a CO ₂ incubator at 37 ° C in an atmosphere containing 5% CO ₂ and 95% air in MEM / F12 medium containing 15% fetal bovine serum, 2 mM L-glutamine, 100 μg / ml gentamicin. The culture medium was changed every 3 days. Reseeding of cells was carried out after the formation of a monolayer in a ratio of 1: 3.

For experiments, cells obtained as a result of the third reseeding were used. The corresponding compounds, including Semax, were added in a volume of 100 μl immediately after the cells were attached to the substrate, once. Assessment of the proliferation of cultured glial cells was performed 24 hours after the addition of the test compound.

The experimental data are presented in figure 2. The results obtained indicate the absence of a significant effect of this peptide on the proliferation of glial cells. Cow embryonic serum was used as a positive control.

Example 4

A study was made of the ability of the Semax peptide to affect the level of expression of neurotrophin genes in primary glia cell cultures. Glia cells isolated as described in Example 2 were seeded after the third reseeding on 4-well plates treated with poly-L-lysine (6 cm well diameter) in 5 ml of DMEM medium containing 15% fetal bovine serum, 2 mM L-glutamine and 100 μg / ml gentamicin, and after the formation of the cell monolayer, the medium was changed to DMEM with 1% serum and 2 mM L-glutamine and 100 μg / ml gentamicin. After 48 hours, 50 μl of a sterile Semax solution was added to the medium to the indicated final concentration or an equivalent volume of sterile water. At the indicated time intervals, the culture medium was taken and the cells were washed with PBS (4 ° C). Total RNA was isolated by phenol-chloroform extraction using the Promega RNAgents ^® Total RNA Isolation System kit (Promega, USA), following the methodology and recommendations of the manufacturer. After ethanol precipitation, the RNA pellet was washed twice with 70% ethanol and stored at -70 ° C. To remove possible impurities of genomic DNA, RNA samples were dissolved in water treated with diethyl pyrocarbonate (DEPC) and incubated for 1 h at 37 ° C in the presence of RNase-free DNase. After additional phenol-chloroform extraction, RNA was precipitated, washed with 70% ethanol, dissolved in DEPC-treated water and stored at -70 ° C.

For reverse transcription, 2 μg of total RNA was selected and the reaction was performed for 1 h at 37 ° C in 25 μl of medium containing 1 μg oligo (dT) ₁₅ primers, 50 mM Tris-HCl, 15 mM (NH ₄ ) ₂ SO ₄ , 5 mM MgCl ₂ , 0.01% gelatin, 0.01% Tween-20, 0.2 mg / ml BSA, 5% glycerol, 2 μM dNTPs, 30 units RNase inhibitor (Fermetas) and 50 U Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Synthol, Russia). After incubation for 10 min at 95 ° C, cDNA samples were frozen and stored at -20 ° C. The obtained cDNA samples were amplified using the following primers and conditions:

These primers are shown in figure 3.

Amplification was performed in a medium with a volume of 20 μl containing 2 μl of cDNA sample, 5 pmol of each primer, one of each pair being labeled with ³² P-αATP using T4 polynucleotide kinase (Sileks, Russia), 5 mM MgCl ₂ , 200 μM dNTPs, 50 mM Tris-HCl, 15 mM (NH ₄ ) ₂ SO ₄ , 5 mM MgCl ₂ , 0.01% gelatin, 0.01% Tween-20, 0.2 mg / ml BSA, 5% glycerol. After initial denaturation for 5 min at 94 ° C, 2.5 U were introduced into the reaction mixture. Taq polymerase (Sileks, Russia) and after 28 cycles (the number of cycles corresponded to a linear dependence of the amount of the product on the amount of the original cDNA) and subsequent incubation for 10 min at 72 ° C, the products were separated in 8% non-denaturing polyacrylamide gel in the presence of ethidium bromide . The bands corresponding to the amplification products were cut out and the radioactivity was determined on a Mark-3 scintillation counter (Nuclear Chicago, USA). All samples were tested in three parallels. The obtained relative amounts of products for NGF and BDNF were normalized by the relative amount of product for β-actin.

The experimental data are presented in figure 4.

The results indicate a significant stimulation of the expression of the NGF (about 5 times) and BDNF (about 8 times) genes under the action of this peptide in primary cultures of glial cells.

Literature

[one]. Freshney R. // Culture of Animal Cells: a Manual of Basic Technique. New York: Wiley-Liss, 1994 .-- 486 p.

[2]. Hefti F., Hartikka J. and J. S.-R. / In: A Dissection and Tissue Culture Manual of the Nervous System. Eds .: A.Shahar and J.de Vellis. - New York: Wiley-Liss, 1989 .-- P.172-182.

[3]. Cole R. and de Vellis J. / In: A Dissection and Tissue Culture Manual of the Nervous System. Eds .: A.Shahar and J.de Vellis. - New York: Wiley-Liss, 1989 .-- P.121-133.

Claims (6)

1. A method for screening pharmacological compounds for neuroprotective activity, comprising culturing primary cultures of neurons derived from a mammalian brain or primary glial cells derived from a mammalian brain, adding a test compound, evaluating the survival of cultured neurons, or evaluating the proliferation of cultured glial cells. 2. The method according to claim 1, where the cultivation of the cells is carried out in a CO ₂ incubator at 37 ° C in an atmosphere containing 5% CO ₂ and 95% air. 3. The method according to claim 1, where the test compound is added once in a volume of 100 μl immediately after the cells are attached to the substrate. 4. The method according to claim 1, where the assessment of the survival of neurons is carried out after 7 days of cultivation. 5. The method according to claim 1, where the assessment of the survival of neurons is carried out after 12 days of cultivation. 6. The method according to claim 1, where the assessment of the proliferation of cultured glial cells is carried out 24 hours after the addition of the test compound.

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