RU2605377C2 - 7-O-[6-O-(4-ACETYL-α-L-RHAMNOPYRANOSYL)-β-D-GLUCOPYRANOSIDO]-5-HYDROXY-6-METHOXY-2-(4-METHOXY-PHENYL)-4H-CHROMONE-4-ON, HAVING ANTI-ALCOHOL ACTION ON HIGHER NERVOUS ACTIVITY - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and concerns use of biologically active flavonoid 7-O-[6-O-(4-acetyl-α-L-rhamnopyranosyl)-β-D-glucopyranosido]-5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-4H-chromone-4-on (acetilpectolinarin - ACP) of formula I, extracted from Linaria vulgaris Mill and showing evident antialcohol action on nervous system. It allows to provide its use as medicinal agent for correction of parameters of behavior: coordination of movements and attention, sober situation assessment, memory - caused by short-time alcoholic intoxication, and chronic alcohol consumption.

EFFECT: technical result consists in implementing the above application.

1 cl, 12 dwg, 2 tbl

formula I

Description

The invention relates to medicine, in particular narcology and neurology, as well as to the pharmaceutical industry and the use of a biologically active flavonoid compound, 7-O- [6-O- (4-acetyl-α-L-ramnopyranosyl) -β-D -glucopyranosido] -5-hydroxy-6-methoxy-2- (4-methoxy-phenyl) -4H-chromon-4-one (acetylpectolinarin-ADC) of the formula

isolated from Linaria vulgaris Mill. and having a pronounced anti-alcoholic effect on the nervous system, which suggests the possibility of its use as a medicine for the treatment of alcohol dependence, as well as for correcting violations of such key behavioral parameters as coordination of movements and attention, sober assessment of the situation, memory, etc. caused by both short-term intoxication and chronic alcohol consumption.

Addiction to alcohol is a significant social and medical problem worldwide. Alcoholism is one of the important causes of serious economic and social problems, starting with disruption of the production process and to damage to the working ability of the world's population. The costs of treating patients, a decrease in labor productivity, an increased risk of somatic and mental illness, early mortality are all consequences of acute and chronic alcohol abuse.

The brain is one of the main targets of alcohol intoxication. The potential costs associated with brain damage from alcohol are enormous. 7-10% of the population of developed countries are diagnosed with alcohol dependence; 9% of them showed clinical brain damage. It has been shown that the brain retains past dysfunctions, even if intoxication is stopped [Special Report to the US Congress: Alcohol and Health, Chapter 2: Alcohol and the Brain: Neuroscience and Neurobehavior; The Neurotoxicity of Alcohol].

Intensive and extensive neurobiological studies have revealed a number of synaptic and extrasynaptic mechanisms, to one degree or another, affected by ethanol. It is known that ethanol is able to interact with lipids and thereby affect the viscosity of cell membranes [Electron paramagnetic resonance studies of ethanol on membrane fluidity / JH Chin, DB Goldstein // Adv Exp Med Biol. 1977; 85A: 111-22]. There is a consensus among researchers that the target of ethanol is GABAergic transmission: either directly by influencing synaptic and extrasynaptic GABA receptors, or by acting on neurosteroids [Ethanol potentiation of GABAergic synaptic transmission may be self-limiting: role of presynaptic GABA (B) receptors / OJ Ariwodola, JL Weiner // J Neurosci. 2004 Nov 24; 24 (47): 10679-86, The effects of acute and chronic ethanol exposure on presynaptic and postsynaptic gamma-aminobutyric acid (GABA) neurotransmission in cultured cortical and hippocampal neurons / RL Fleming, PB Morris, AL Morrow // Alcohol. 2009 Dec; 43 (8): 603-18. doi: 10.1016 / j. alcohol.2009.10.00.006, Brain steroidogenesis mediates ethanol modulation of GABAA receptor activity in rat hippocampus / E Sanna, G Talani, F Busonero, MG Pisu, RH Purdy M Serra G Biggio // J Neurosci. 2004 Jul 21; 24 (29): 6521-30, Effects of ethanol on GABA (A) receptors in GABAergic and glutamatergic presynaptic nerve terminals / M Wakita, MC Shin, S Iwata, N Akaike // J Pharmacol Exp Ther. 2012 Jun; 341 (3): 809-19. doi: 10.1124 / jpet. 111.189126. Epub 2012 Mar 20]. At the same time, a satisfactory explanation of the stimulating and inhibiting effect of alcohol on the psyche has not been received.

Despite intensive studies of possible molecular mechanisms, behavioral effects, there are still no medications that have a therapeutic effect on alcohol intoxication of the brain and nervous system. There is not enough information on the effect of ethanol on the behavior of small neural networks isolated from the rest of the brain. This test system demonstrates the effect of a substance on a local network without involving the concomitant effects of external afferents or the action of neurons external to this network.

University of California scientists have found in animal studies that Dihydromyricetin (DHM), isolated from Hovenia dulcis Thub., Counteracts acute alcohol intoxication by blocking the effects of alcohol on the brain, reducing the addiction to voluntary use by animals and helping to relieve symptoms of a hangover. DHM has been shown to eliminate the effect of alcohol on inhibitory GABA (A) receptors of the brain [Dihydromyricetin As a Novel Anti-Alcohol Intoxication Medication / Yi Shen, A. Kerstin Lindemeyer, Claudia Gonzalez, Xuesi M. Shao, Igor Spigelman, Richard W. Olsen, Jing Liang // The Journal of Neuroscience, January 4, 2012 - 32 (1) - S. 390-401].

This study should be taken as a prototype due to a similar class of chemical substance and type of biological activity. The disadvantage of the prototype are the following provisions.

1. The established anti-GABAergic activity of DHM characterizes its effect only in case of intoxication with high doses of alcohol.

2. The study lacks data on the effect of alcohol on the neural activity of networks in the field of physiological concentrations of 0.09-0.5%. The alcohol content in the blood of humans and experimental animals at concentrations of the order of 0.5% and higher are lethal and are not of practical interest.

3. There are no studies to establish a possible mechanism for the action of physiological concentrations of alcohol on the activity of neural networks.

4. There are no studies on the effect of DHM on chronic alcohol intoxication.

5. Limited distribution of the producing plant. Homeland Hovenia dulcis Thub. - China; bred in India (Himalayas), Japan. In Russia and neighboring countries they cultivate in the warm regions of the Caucasus, Central Asia and Crimea (sometimes the shoots freeze) [Official site of the dictionary “The Great Soviet Encyclopedia”, http://www.big-soviet.ru/ (accessed April 22, 2014)] .

The proposed invention differs from the prototype in that the biologically active substance of the ADC isolated from Linaria vulgaris Mill begins to act even at low and medium levels of alcohol intoxication. The flavonoid ADC compound is a “mild” selective SK-channel inhibitor, eliminates the direct neurotoxic effect of alcohol on the brain and corrects abnormal behavior caused by both short-term intoxication and its chronic use. It has been established that small potassium (small potassium, SK) channels are the target of the entire spectrum of physiological concentrations of ethanol. In addition, the producing plant Linaria vulgaris Mill. widely distributed in Russia and neighboring countries. The raw material base of the plant is sufficient for the procurement of raw materials on an industrial scale.

Previously, in a few studies, it was found that ADC has a cardiotonic effect, increases the amplitude of heart contractions and slows their rhythm, with an increase in dose it causes a hypotensive effect, accompanied by increased and deepened breathing [Distribution of common flax in the USSR and the content of acetylpectolinarin and pectolinarin / A in it .AT. Patudin, B.A. Krivut, L.S. Demidova // The First Republican Conference on Medical Botany: Abstracts. - Kiev, 1984 .; To the pharmacological characteristic of acetylpectolinarin / M.I. Rabinovich, A.R. Zeltser // Transactions of the Trinity Veterinary Institute, 1970. - Vol. 13, no. 1. - S. 69-73 .; The Norichnikov family - a source of obtaining new plants of cardiovascular action / M.I. Rabinovich, A.I. Schröter, A.V. Ermolin and others // Proceedings of the Trinity Veterinary Institute, 1970. - T. 13, issue. 1. - S. 74-81]. Data on the anti-alcohol activity of ADC in the literature was not found.

Flavonoid ADC isolated from Linaria vulgaris Mill. Refers to low-toxic compounds. LD ₅₀ with subcutaneous administration was 825 mg / kg, with long-term administration of the ADC for 30 days, deviations in the condition of the experimental animals were not found, after opening of pathological changes in the internal organs was not noted [To the pharmacological characteristics of acetylpectolinarin / M.I. Rabinovich, A.R. Zeltser // Transactions of the Trinity Veterinary Institute, 1970. - Vol. 13, no. 1. - S. 69-73].

The aim of the invention is the establishment and description of a highly active compound of plant origin with a sufficient raw material base of the producing plant, acting at different levels and with different durations of alcohol intoxication, eliminating the direct neurotoxic effect of alcohol on the brain and correcting the abnormal behavior caused by its single and chronic use.

This goal is achieved by isolating the ADC from the aerial part of Linaria vulgaris Mill. [Smirnova, L.P. Chemical study of flavonoids of some species of flax and sage: dis. ... cand. Pharmac Sciences: 15.00.03: it is protected 04/26/76 / Smirnova Liliya Porfirevna. - Moscow, 1976. - S. 53-64].

The method of obtaining the ADC. Plant material 50 g is crushed to a particle size of not more than 2 mm, extracted with 95% ethanol 400 ml each by heating in a water bath under reflux for 2 hours three times. The resulting extracts are combined, dried under vacuum. The dry residue is dissolved in a minimum amount of dioxane-water mixture (1: 1) and mixed with 10 g of silica gel. The resulting powder is dried at a temperature of 40 ° C, applied to a chromatographic column with a diameter of 30 mm, prepared from 40 g of silica gel. The column was washed with 1 L of carbon tetrachloride, and then eluted with chloroform-methanol (96: 4). The eluates were collected in 200 ml portions and examined by TLC in a solvent system of ethyl acetate-ethanol-water (100: 27: 13). The eluates having on the chromatograms after treatment with a 5% alcohol solution of aluminum chloride one yellow adsorption zone with a value of R _f = 0.68 ± 0.01 are combined. The eluent is distilled off under vacuum, the residue is dried at a temperature of 40 ° C, crystallized from ethanol. The yield of the finished product is 1.8% by weight of plant materials. _Mp = 242-244 ° C.

The inventive compound is a light yellow crystalline substance, soluble in ethanol, when heated in a water bath to 30 ° C, readily soluble in chloroform. When dissolved, it forms turbid solutions. It is very easily soluble in dimethyl sulfoxide and 1,4-dioxane to form a clear solution. It is practically insoluble in water. In the PMR spectrum (Bruker-300 (300 MHz) in DMSO-d ₆ , internal standard is TMS) of compound I there are: proton signals — OCH ₃ groups (3.78, 3.86 ppm), proton signals of a para-substituted aromatic ring ( two doublets at δ = 7.15 ppm and δ = 8.05 ppm with J = 8.9 Hz), the proton signal —COCH ₃ (singlet at δ = 1.97 ppm), the proton signal of the OH group of the flavon (singlet at δ = 12.94 ppm), proton signals at the third and eighth carbon atoms (singlets at δ = 6.99 ppm and δ = 6.41 ppm, respectively), signal -CH ₃ groups (doublet at δ = 0.86 with J = 6.3 Hz), signals of ten methane protons, one methylene group and five hydro yl groups in the range of 5.50 ppm up to 3.20 ppm

The technique of preparing a neural culture. The influence of ADC on cell activity against the background of alcohol was performed on a neuron culture made according to the generally accepted protocol [Functional plasticity triggers formation and pruning of dendritic spines in cultured hippocampal networks / M Goldin, M Segal, E Avignone // J Neurosci. 2001 Jan 1; 21 (1): 186-93]. The pups underwent rapid decapitation directly on their birthday (P0), after which their brain was quickly withdrawn and placed in a cooled (up to 4 ° C), oxygenated Leibovitz L15 solution (from Gibco), enriched with glucose in the amount of 0.6% and antibiotic - gentamicin (Sigma, at a rate of 20 μg / ml). The hippocampal tissue underwent mechanical dissociation after incubation in a solution supplemented with trypsin (0.25%) and deoxyribonuclease (50 μg / ml) and transferred to an incubation solution containing 5% inactivated horse serum (HS), 5% fetal calf serum and complex factor growth of B-27 (1 μl / 1 ml), based on the minimum basic medium (minimum essential medium, MEM), manufactured according to Earl's protocol (Gibco), enriched with 0.6% glucose solution, gentamicin (20 μg / ml), and 2 mm GlutaMax (Gibco) (the so-called enriched MEM).

Approximately 10 ⁵ cells in 1 ml of medium were plated in each well of a 24-well plate, over a layer of hippocampal glial cells, seeded and grown on 13 mm coverslip 2 weeks before neurons were introduced [Morphological analysis of dendritic spine development in primary cultures of hippocampal neurons / M Papa, MC Bundman, V Greenberg, M Segal // J Neurosci. 1995 Jan; 15 (l Pt 1): 1-11]. Cells were placed for growth and development in a sterile incubator at a temperature of 37 ° C in an atmosphere containing 5% carbon dioxide for four days. After this, the medium was changed to 10% HS in enriched MEM with the addition of a mixture of 5-fluoro-2′-deoxyuridine (5-fluoro-2-deoxyuridine / uridine, FUDR) (Sigma; 20 μg and 50 μg / ml, respectively) for blocking glial proliferation. Four days later, this solution was replaced with 10% HS in MEM and remained unchanged until the day the culture was used in the experiment.

Technique of electrophysiological registration. A neural culture was placed for physiological registration in the flow chamber. Its positioning relative to the microscope in the X / Y planes was carried out by a motorized platform manufactured by Luigs & Neuman (Germany). Cells were placed on the stage of a Zeiss confocal microscope - PASCAL LSM-5. Used water-immersion lenses: Olympus x63 (0.9NA) or Zeiss x40 (0.8NA). To review the cells and scan them, one of the lenses was immersed directly in the registration washing solution. The composition of the standard solution for registration: NaCl (129 mm), KCl (4 mm), MgCl ₂ (1 mm), CaCl ₂ (2 mm), glucose (10 mm), HEPES (10 mm). pH = 7.4 is established by adding NaOH, osmolarity at a level of about 320 mOsm - using sucrose.

Neuronal activity was recorded using the patch clamp method using glass micropipettes containing a solution for intracellular registration: K-gluconate or CsCl (140 mM), NaCl (2 mM), HEPES (10 mM), Na-GTP (0.3 mM), Mg-ATP (2 mM), phosphocreatine (10 mM), pH 7.4. The current resistance for the pipettes was selected at the level of 6-10 mOhm. The signals were amplified using an Axopatch 200A amplifier (Axon Instruments), and stored for computer processing. Data was analyzed using pCLAMP software (MolecularDevices).

Visualization method for intracellular calcium. The culture was incubated for one hour at room temperature and in the above registration solution containing 3 μM Fluo-2HA (AM) calcium sensor. Due to the passive unilateral penetration of this sensor into the cells, its concentration there reached values of about 50 μM, which is sufficient for reliable fluorescence recording of intracellular calcium signals. After the end of the incubation period, the cells on the glass were transferred to an identical solution without dye and fixed in a recording flow chamber on the stage of one of the confocal microscopes: direct Zeiss PASCAL LSM-5 (Germany) equipped with Olympus x63 water-immersion lenses (0.9NA ) or inverted Zeiss LSM-510 (Germany) with an oil-immersion lens Zeiss x40 (1.3NA). In the second case, a motorized table made it possible to fix, store in the computer memory and reproduce the location of several recording fields within the same preparation. The flow-through perfusion system was built on the basis of a passive inflow from a solution source on an elevated platform - sterile 30 ml disposable syringes. Outflow was carried out by forced excess suction with an adjustable level in height on the basis of a peristaltic pump with a variable speed of rotation. The pinhole was installed in the closed position, allowing to obtain optical sections with a thickness of 3-5 μm using an acousto-optical modulator. This experimental system made it possible to halve the concentration of the calcium sensor (which reduced the appearance of artifacts) while maintaining the quality of visualization. Images were formed by scanning at high speed and resolution in fluorescence exciting light of an argon laser at a light wavelength of 488 nm and a radiation spectrum of about 505-550 nm. High scanning speed was achieved by the method of bidirectional mirror movement, as well as scanning of limited areas of the field of view (the so-called “regions of interest”) with a reduced total number of pixels. The total scanning speed reached 50 ms / frame or less, which is 20 Hz or more. A series of images lasting from several minutes to several tens of minutes (usually from 5 to 40 minutes), depending on the size of the current frame in pixels and on the nature of the experiment, was written onto the stack. Series of images were pre-processed using Zeiss LSM, Image J and Kaleida Graph software.

Investigation of the interaction of ADC and ethanol in vivo and in vitro. The in vitro effect of ADC on cell activity against the background of a wide range of alcohol concentrations (from 0.1 to 4% in washer saline) was carried out in a primary culture of rat hippocampal neurons; the in vivo effect on the correction of behavior in animals under the influence of physiological concentrations of alcohol (with blood concentrations ranging from 0.01% -0.5%) was performed in laboratory mice and rats.

Estimation of alcohol concentration in the experiment. It should be noted that the concentration of alcohol in the blood, after drinking it in the form of alcoholic beverages, reaches values of 2-3 ppm (0.2-0.3%) with light and medium levels of intoxication and about 5 ppm (0.5%) with severe intoxication. A further increase in blood alcohol is considered incompatible with life [A hybridizing of effects as described at Alcohol′s Effects from Virginia Tech and Federal Aviation Regulation (CFR) 91.17: Alcohol and Flying]. For humans (men and women weighing about 70 kg), as well as for laboratory rats (Wistar breed weighing about 100 g), there is the following ratio of the amount of ethanol consumed (EtOH) and its concentration in the blood, measured at half-hour intervals:

The amount of ethanol in the blood is related to its concentration in the brain. For humans, there is no formula for a simple correlation between the ethanol content in the blood and brain tissue. However, using experimental data with rats of various breeds, with a parallel measurement of the level of EtOH in the blood and in the brain [M. Nurmi, K. Kllanmaa, J.D. Sinklair, Brain ethanol levels after voluntary ethanol drinking in AA and Wistar rats, Alcohol, 19, 2, 113-118, 1999], found that the concentration of ethanol in the brain over the next 5-15 minutes after ingestion exceeded those in the blood of 1.5 times, and with its rapid admission - 3 times.

Thus, physiological concentrations of ethanol in the brain cannot exceed 1.5-2%. It is assumed that ethanol freely overcomes the blood-brain barrier and enters the brain, where it accumulates to the specified limit due to affinity properties. Thus, the 4% ethanol concentration used in in vitro experiments, when complete inhibition of neuronal activity is observed, cannot be considered physiologically significant, while the 0.25-1.5% region in the brain tissue is physiological and is of practical interest.

Statistical analysis. Zones corresponding to individual neurons within the field were averaged over the gray level and transformed into digital matrices. The records were assigned a unique code number, and the data of the entire optical field was stored for further processing. The analysis of digital data obtained from optical stacks was performed using the software system created by the authors in a high-level programming environment MatLab to solve a number of problems ¹ ( ¹ Compensation of the effect of dye fading, noticeable during long recordings. Separation of the recording into separate sections in accordance with the pharmacological effect. Normalization of various records according to the scanning frequency, depending on the size of the optical field.Automatic detection of spikes according to the criterion developed by the authors, in comparison with din the standard deviation of the noise level, calculating the number of spikes per unit time, measuring the average amplitude of spikes, transforming records using the ΔF / F principle (subtracting the average value of the "background" and dividing by this value). Calculating the average spike based on all recorded spikes. Definitions coefficient of synchronization between individual cells in the studied fields (1 - maximum; 0 - minimum). All types of calculations were accompanied by statistical analysis: determination of variance, standard deviation, dispersion ANOVA analysis, as well as retrospective or post-hoc.).

The results were displayed in the form of graphs, tables and separate digital matrices. Some of them were transferred to KaleidaGraph and Photoshop for final processing and creating illustrations.

Electrophysiological data were studied using pClamp 8 software. Statistical analysis was performed using T-test methods (Student's test), ANOVA and Wilcoxon-Mann-Whitney. In some cases, the programming environment MatLab was used.

1. In vitro studies

Description of the background impulse activity of neurons. The spontaneous activity of neural networks in the culture was stable for 40 minutes of recording (Fig. 1A). Examples (frame-insets) of the interspike period, peak, and also the stage of spike development at three time points are shown (Fig. 1B). Net rasters indicate images with a subtracted background level. The figure shows the different dynamics of the development of spikes in different cells of the network during their general synchronization. The number of spikes, their dynamics (shape), amplitude and field synchronization coefficient remained unchanged (Fig. 1B-E, respectively).

Thus, background activity is stable enough for pharmacological studies.

The effect of ethanol on neural activity. In total, the averaged results from 27 experiments covering 162 neurons were used. During continuous recording of intracellular calcium levels, increasing concentrations of ethanol were added (Fig. 2). It was found that low concentrations of 0.5% (or ~ 85 mm) and 1% (~ 170 mm) cause a significant increase in the frequency of spikes in cultured neurons (a series of 5 glasses, a total of 34 cells; 95% increase in the frequency of discharges: from 13.5 ± 1.45 to 26.4 ± 1.84 spikes in 100 seconds, p <0.0004, according to the paired T-test). This increase is associated with a shortening of the spike duration (1/2 decay = 0.442 ± 0.065 sec in the control, versus 0.319 ± 0.041 sec in the presence of 0.5% ethanol, p <0.0001) (Fig. 2A, examples 1-3 and B). In addition, a low concentration of ethanol caused an increase in the coefficient of synchronization between cells (Fig. 2G). High ethanol concentrations (2-4%), on the contrary, significantly reduced the frequency of discharges until the spontaneous activity was completely suppressed. In the process of washing ethanol, a gradual recovery to normal was observed.

In a series of experiments (Fig. 2B), when testing an even lower concentration of ethanol (0.25% or about 40 mM), an increase in activity at low concentrations (0.25-0.5%) and its gradual decline at high concentrations (1-4%) were found. The minimum concentration of ethanol, capable of causing a statistically significant increase in impulsation, was 0.1% (about 15 mm). Washing ethanol returned to the initial level both the frequency of the spike discharges and the synchronization coefficient between them.

Thus, in the effect of ethanol on neural activity, the activation phase (at low and medium physiological concentrations in terms of the level in the brain: 0.1-1%) and the inhibition phase (at high physiological concentrations of more than 1% in terms of the level in the brain, as well as at transcendental concentrations) are distinguished more than 2%, which can be created only in vitro, but not in vivo, as they lead to inhibition of the center of respiration and death).

Chronic effect of ethanol on cultured neural networks. The effect of ethanol at a concentration of 1% was studied on a hippocampal culture of neurons after 0.5, 1, 2, and 5 days of its continuous exposure. The stability of the concentration of ethanol in the solution was controlled. The spontaneous activity of neurons was recorded after removal of ethanol and compared with the control. It was found that a relatively short stay of ethanol in neuronal culture (12-24 hours) significantly increases the level of spontaneous activity measured in 100 seconds: 14.3 ± 1.18 in the control, 21.8 ± 2.27 after 0.5 days and 24.57 ± 2.51 after 1 day (p < 0.0001). After 48 hours, the activity level approached the control and did not statistically differ from it (p> 0.43), and after 5 days it decreased significantly below the control: 5.37 ± 0.66 (p <0.0001).

Thus, both in acute and chronic exposure to ethanol, a phase of pathologically high impulse in neural networks has been established, which gradually passes into the phase of abnormal inhibition.

Given this fact, the therapeutic effect on alcohol intoxication of the brain should be based on the prevention of these symptoms, as well as on the elimination of symptoms that have already arisen.

The effect of ADC on neural activity in the control and on the background of ethanol (EtOH). In order to counter the pathological effect of ethanol on the impulse activity of neurons, the biologically active substance of the flavonoid nature of the ADC was first tested. It was found that at a dose of 1 to 30 μM, the ADC practically does not change the impulse pattern. A decrease in activity was observed at 50 μM and especially 80 μM (Fig. 3A). Dosages of 60 μM and higher significantly reduce impulses; frequency data summarizes the study on 134 cells (Fig. 3B). The amplitude also changes in a similar way; it decreases after adding an ADC at a dose of 60 μM and significantly decreases at a dose of 120 μM (Fig. 3B). Washing effectively returns activity to normal, indicating the non-toxic nature of the changes. Of particular interest is the dynamic effect of the flavonoid compound. The results presented in Figure 3G, D prove that at the beginning there is a short-term, statistically significant phase of the increase, after which the activity of neurons returns to normal (Fig. 3G); the amplitude of the spikes during the period of increase insignificantly and briefly decreases (D).

The impact of ADC at a dose of 10 μM on a neural culture during 7 days of incubation did not lead to a decrease in the number of neurons compared with the control (p> 0.45), which indicates the absence of neurotoxicity of the ADC with long-term (chronic) administration.

The key is the effect of the flavonoid on neural activity, which changed under the influence of ethanol. It was found that the ADC prevents the excitatory phase of the reaction to ethanol: 8.75 ± 1.15 in the control; 9.21 ± 0.95 in 10 μM ADC; 7.9 ± 1.73 in ADC + 0.5% EtOH; and 7.5 ± 1.5 in ADC + 1% EtOH (33 experiments, n = 168 cells). In the event that the EtOH effect has already developed, the introduction of the ADC returned the impulse to normal: 8.46 ± 0.98 in the control; 14.31 ± 1.71 in the presence of EtOH; and 9.34 ± 1.12 after adding ADC (24 experiments, 122 cells) (Fig. 4A, B). The comparative effect of different doses of ADC against ethanol is shown in Figure 4. It is shown that the introduction of 10 μM ADC restores the increased level of impulse to normal, while a dose of 100 μM reduces it (Fig. 4B) and also reduces the amplitude of the spikes (C). These effects are successfully eliminated by washing.

Thus, therapeutic doses should be recognized as ADC doses not exceeding 30-60 μM, since ADC in the indicated dosages reliably eliminates or prevents the pathological effect of ethanol. High doses of ADC reduce the impulse caused by ethanol, but the effect is reversible, indicating a lack of neurotoxicity.

The effect of ADC on cultured neural networks in the chronic action of ethanol. The presence of 10 μM ADC in the nutrient solution effectively prevented the pathological effect of ethanol caused by its long-term administration. A pronounced increase (up to 2 days of incubation) and decrease (5 days of incubation) of the impulse activity of neurons was established. The spontaneous activity of cells at all periods of testing, measured after elimination of both ADC and ethanol, did not statistically differ from that in the control group (14.3 ± 1.18 in the control; 15.9 ± 2.56 every other day; and 12.6 ± 3.08 5 days after chronic testing, p> 0.72).

Thus, the ADC blocks the pathological effect of ethanol in the neural culture of the hippocampus caused by its long-term (chronic) administration.

Analysis of the mechanisms of the action of ethanol in vitro on the activity of neural networks

The study of synaptic currents on the background of ethanol. The study was conducted according to the method of electrophysiological fixation of the membrane locus. Tetrodotoxin (1 μM) was used to block spikes, and bicucullin (10 μM) was used to suppress GABAergic activity. Under these conditions, miniature postsynaptic currents (mVPST) were recorded in the control and after adding ethanol at a concentration of 0.5 and 3% (Fig. 5).

Analysis of the obtained data showed that in the latter case, the amplitude of mVPCT significantly decreased compared to the control (Fig. 5B). The number of spontaneous events increased with the addition of ethanol (Fig. 5G, D). Significant increase in frequency (p <0.05). It should be noted that the presence of bicucullin did not allow inhibitory postsynaptic potentials to develop, whereas in its absence, ethanol at a concentration of 3% caused a suppression of activity (see Fig. 2). It was found that the growth phase of mVPST was significantly shorter in the presence of ethanol (Fig. 5E). The change in dynamics was quantitatively small, but the differences were statistically significant (p <0.01).

The totality of the data indicates the presence of two phases, namely, excitatory and inhibitory, under the action of ethanol. Moreover, inhibition is observed at a high physiologically unrelevant concentration (3%) and “masks” the excitatory component. In addition, the shortening of the growth phase of mVPCT is probably associated with the involvement of voltage-dependent potassium channels in the excitatory effects of ethanol.

Thus, the effect of ethanol on neural activity is mixed - excitatory and inhibitory (in this case, the excitatory effect is probably associated with the action of voltage-dependent channels).

The inhibition effect is investigated in the next series of experiments. The effect of ethanol on neuroactivity during blockade of inhibition. In accordance with the previously established paradigm, after the addition of 0.5% ethanol, the excitation phase is observed, and after 4%, the phase of inhibition of activity is observed (Fig. 6A). Under the influence of bicucullin, a blockade of the GABAergic component occurs; adhesions become longer and higher in amplitude (Fig. 6A, examples 1 and 2). Against this background, the addition of 0.5% ethanol speeds up, and 4% no longer reduces spontaneous activity, systematically narrowing the width of the spikes (Fig. 6A, examples 3 and 4). The general picture summarizing the five experiments, frequencies and amplitudes is shown in Figure 6B, C. Panel D allows you to compare the effects of 4% ethanol in the presence and absence of bicucullin. Ethanol at a concentration of 0.5% in both cases increased activity, but at a concentration of 4%, it no longer suppressed impulses in the presence of a GABA antagonist.

Thus, the effect of inhibition of ethanol is due to increased GABAergic activity. In the field of physiological concentrations (0.5-1.5%), the effects of excitation and inhibition of ethanol overlap.

The channel of the excitatory effect of ethanol on neural activity. The question of how ethanol acts on the nervous system is of fundamental importance. The presence of a connection between ethanol and potassium channels was found in a study using a specific SK-channel blocker, apamine toxin. Figure 7A shows the dynamics of the effect of apamine (40-60 nM) on pulsation. The first phase of short-term activation is followed by stabilization of neuronal activity at a slightly elevated or normal level with increased discharge amplitude (Fig. 7A, D). The addition of ethanol (0.5-1.5%) against the background of apamine did not lead to a further increase in impulsation, however, ethanol at higher concentrations still depressed it, and washing washed back to normal. Also, the shortened spike dynamics caused by ethanol was restored by apamine to normal (Fig. 7B). A comparison of the effect of ethanol at a concentration of 0.5% with and without apamine is shown in Figure 7G. Apamine causes a short-term increase in frequencies, but blocks the effect of ethanol (data from nine experiments are summarized - 44 cells).

Similarly, against the background of the excitatory effect of ethanol, the introduction of apamine (40 nM) returned impulse to normal (Fig. 7E, 3). In this case, however, as in the first case, the amplitude of spike discharges increased (Fig. 7G). It should be noted that the long-term (chronic) effect of apamine on a neural culture was accompanied by a neurotoxic effect (27.3% ± 9.165 cell death compared with the control), which is probably associated with an increased pulse amplitude.

Thus, the exciting effect of ethanol is due to the participation of voltage-dependent potassium channels of the SK type.

The mechanism of action of ADC on neural activity. The biologically active substance of the ADC, with the identical experimental strategy (see above), similarly to apamine, affected the pathological excitation provoked by 0.5% ethanol (Fig. 7I, K). ADC at a dose of 10 μm blocks the excitatory effect of ethanol on impulses (114 cells), while causing a short-term increase in the spontaneous activity of neurons. After 10 minutes, it completely returns to the control level, after which the effect of ethanol is blocked.

The almost complete coincidence of the picture is evident both in the case of the introduction of the ADC against the background of ethanol (I), and when ethanol is fed against the background of the ADC (K). In both cases, the excitatory effect of ethanol was completely blocked. The excitatory effect of the ADC itself was short-term and identical to the effect of apamine. However, the principal difference between the ADC effect and apamine was the absence of an increase in the spike amplitude (Fig. 3) and signs of neurotoxicity.

Like apamine, ADC did not affect the effects of higher ethanol concentrations. The data are shown in Figure 8. As shown above, ethanol at a concentration of 3% significantly reduced impulse in neural networks. Different doses of ADC from 5 to 100 μM did not compensate for this inhibitory effect of ethanol, i.e. did not increase the pulsation (Fig. 8A) and the amplitude of the spikes (Fig. 8B).

The ADC operation was proved by acting on SK channels using a selective SK channel agonist, 1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (1-EBIO) (Fig. 9). Its effect on SK channels depends on concentration: at the beginning, this agonist, like ethanol, enhances neural impulses (Fig. 9A). Then, as the concentration increases (from 200 nM to 500 μM), the pulsation begins to be suppressed and finally disappears. This effect is not associated with an increase in the GABAergic system, since it is not blocked by bicucullin [Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels / E. Korkotian, T. Bombela; T. Odegova; P. Zubov; M. Segal / PLOS ONE, 2013 .-- N. 8, - Is. eleven.]. Thus, the action of 1-EBIO coincided with the action of ethanol only in its initial phase, but not in the inhibition phase. The latter is due to the fact that after the opening of most or all of the SK channels, the cells lost their ability to depolarize. As expected, the 1-EBIO effect was successfully eliminated by a specific SK-channel blocker - apamine (Fig. 9A, B). It was found that ADC acted in a similar way, within therapeutic doses it restores spontaneous activity, which proves the selective and specific nature of its effect on the culture of neurons (Fig. 9B).

Thus, the biologically active flavonoid ADC compound in low and medium doses (from 1 to 30 μM) briefly enhances the impulse without changing its general pattern. High doses of ADC (> 80 μM) suppress impulses. The effects of ADC are reversible with short-term and long-term exposure, which indicates the absence of neurotoxicity. ADC does not affect the balance of inhibition in neural networks, but it has a pronounced anti-alcohol effect in the field of physiological concentrations of ethanol, eliminating the phase of the pathological increase in impulse. It is proved that the action of the ADC is mediated by a non-toxic and reversible effect on the SK channels, which is expressed in the selective decrease in conductivity in this type of potassium channels.

2. In vivo studies

1. The test "cross the maze." The cross-shaped labyrinth consists of 4 chambers (numbered 1, 2, 3, 4), interconnected through the same fifth central chamber. The dead end and the central chamber of the labyrinth for mice are a cube with an edge of 15 cm long and an entrance hole with a cross section of 7 × cm. In the labyrinth for rats, the central chamber is a cube with an edge of 20 cm long, and the dead ends are 30 × 20 × 20 cm and are connected to the central chamber by a 12 × 12 cm entry hole.

The animal was placed in the central chamber, the labyrinth was closed with a transparent lid on top and allowed to examine the apparatus premises until the animal made 13 visits to its dead-end chambers. A dead end is considered valid if the animal transfers all 4 legs to this section of the maze.

Interpretation of the results. The sequence of transitions and their duration is recorded using a personal computer program. Subsequent analysis is carried out using a program that identifies and evaluates the following types of behavior.

- The total time in the maze spent on 13 visits to its dead ends, which is a correlate of research activity in the open field test.

- The latent period of the beginning of the labyrinth study is the time between the placement of the animal in the central chamber and its first entry into the dead end. This indicator correlates with the avoidance of visiting the open arms of the elevated plus-shaped labyrinth and is proposed for the selection of anxiolytic effects of substances.

- A complete tour of the labyrinth, continuing until the moment when all its compartments are visited. This behavior reflects the ability for spatial orientation and is considered as one of the forms of elementary rational activity in animals. The more effective the spatial orientation, the lower the number of visits spent on the first full round, and the greater the total number of complete full rounds.

- Return to the dead end visited at the previous visit. This behavior is considered as an indicator of short-term memory errors.

- A visit to two of the four dead ends, which is interpreted as behavioral stereotype. Such an episode of behavior occurs if three out of four bays were visited in a row for three or more visits to dead ends in a row.

- The number of right and left turns when moving from a dead end to a dead end through the central compartment of the maze, reflecting the degree of asymmetry of locomotion.

The conditions of the experiment. The study was performed on non-linear white mice of both sexes.

1. Control group: sterile saline was injected intraperitoneally.

2. Ethanol group: ethyl alcohol was injected in the amount necessary to create a concentration in the blood in the range of 0.01% -0.5%.

3. Ethanol-flavonoid group: an ADC solution was injected in an amount of 10 mg / kg and after 10 min ethyl alcohol was injected in an amount necessary to create a concentration in the range of 0.01% -0.5% in the blood.

Results. In alcoholized animals, a decrease in the latent period, total time in the labyrinth, and time of complete bypass compared with the control was noted in the cruciform labyrinth test, which confirms the increase in activity established in experiments on hippocampal cells. In addition, a pronounced stereotype of behavior and asymmetry of locomotion were observed in comparison with the control group of animals.

In animals that were previously intraperitoneally injected with an ADC solution, an increase in the total time spent in the labyrinth compared with alcoholized animals was observed, as well as the time spent by the animals to complete a complete bypass, which indicates the prevention of the occurrence of spontaneous activity of nerve cells caused by the action of alcohol.

2. Test "rotating rod". The installation is a rod 6 cm in diameter, raised to a height of 60 cm, with a fixed rotation speed of 4.0 to 40.0 rpm. When studying the effect of pharmacological substances on alcohol intoxication, mice are pre-tested to achieve stable values (retention time on the rod).

Interpretation of the results. The ability of animals to maintain equilibrium and increase the residence time on a rotating rod under the action of the studied substance, introduced against the background of ethanol, is considered as the ability to reduce the toxic effect of ethanol and restore coordination of movement.

The conditions of the experiment. The rotation speed of the rod is 10 rpm, male mice weighing 25-35 g.

1. Control group: sterile saline was injected intraperitoneally.

2. Ethanol group: 12% ethyl alcohol was injected in the amount necessary to create a concentration of 0.05, 0.1, 0.2, 0.3, 0.5% in the blood. For male white mice weighing about 25 g, the following correlation is known [YOU Charts by Virginia Tech] the following ratio between the amount of 95% ethanol taken and its blood content with the intraperitoneal route of administration.

3. Ethanol-flavonoid group: ADC solution was injected in an amount of 10 mg / kg, and after 10 minutes ethyl alcohol was injected in the amount necessary to create a blood concentration of 0.3%. Estimated retention time of mice on a rotating rod.

As follows from the data obtained (Fig. 10), the behavior of mice with the introduction of ethyl alcohol at a concentration of 0.05% corresponds to the control group. With an increase in the concentration of ethanol in the blood to 0.1 and 0.2%, part of the mice becomes more active (they climb over partitions on the rod, as a result of which they fall). An ethanol concentration of 0.3% is the most acceptable; animals have a state of agitation and muscle weakness (mice are active, but there is no coordination of movements). With the introduction of ethanol in a dose of 0.5%, the mice are motionless (sleep). With intraperitoneal administration of ADC, the behavior of animals corresponds to the control group.

The difference between the results of the control group and the results of the ethanol group is statistically significant (*, T-test).

Results. In alcoholized animals, a state of increased activity was noted in the test of a rotating rod, and a change in coordination of movements was observed in comparison with the control group.

It was found that in the time interval from 10 to 40 minutes after intraperitoneal administration of the ADC against the background of alcohol, the time spent by animals on the rod significantly increases, which indicates the restoration of coordination of movements (Fig. 11).

3. Test "open field". The study was conducted on rats of different ages (from 5 to 9 weeks). Separately studied the behavior of males and females. The open field is a white laminated square box with a side of 1 meter and a height of 50 cm, open at the top. The base of the box is drawn into squares with a side of 25 cm, so there are 16 squares in total. The animal can freely move along the surface of the box, crossing the lines of the inner squares. An experiment with each animal is carried out for 3 minutes. The animal is placed in one of the corner squares and at the same time the timer starts. The total number of crossed lines in 3 minutes is fixed automatically.

Interpretation of the results. Control animals instinctively try to avoid moving in open space, so the average number of lines crossed by them is small. The effect of ethanol on the consciousness of an animal can be expressed in disinhibited behavior, partial or complete loss of fear of moving through open space.

The conditions of the experiment.

1. Control group: sterile saline was injected intraperitoneally.

2. Ethanol group: 30% ethanol solution was injected in saline, intraperitoneally. The volume of injected solution was calculated based on the weight of the animal and was controlled by the actual level in the blood. The experiment was carried out after 30 minutes after injection.

3. Ethanol-flavonoid group: a 30% solution of ethanol in physiological saline was injected (volume as in paragraph 2) and after 5 minutes an ADC solution was injected at a concentration of 3, 5 and 10 mg / kg of animal weight. The experiment was carried out after 30 minutes after injection of ethanol.

Results. The average number of crossed lines in the control group (6 animals at the age of 4 weeks, 4 animals at the age of 5 weeks, 4 animals at the age of 9 weeks) did not depend on the age of the animals and amounted to 8.2 ± 3.6. After the introduction of low concentration ethanol (0.1–0.15%), the number of crossed lines increased sharply — to 22.1 ± 3.2. In addition, a more pronounced effect of ethanol on the group of young rats (5 weeks) was found compared with the older group (9 weeks). High ethanol concentrations suppressed the motor activity of animals, and the number of crossed lines decreased with respect to the control down to zero. It was found that the ADC at a dose of 5-10 mg / kg blocked the activating effect of only low ethanol concentrations, reducing the number of crossed lines to 10.7 ± 4.6 (Fig. 12). Moreover, ADC alone did not significantly affect the behavior of rats in this test, nor did it affect the effects of high ethanol concentrations.

Thus, it was found that ADC effectively blocks the effect of low concentrations of ethanol in the blood of rats, associated with pathologically disinhibited behavior and loss of natural caution.

The difference between the results of the control group and the results of the ethanol group is statistically significant (***, T-test). The difference between the results of the ethanol-flavonoid group and the results of the ethanol group is statistically significant (**, T-test), the difference between the results of the control group and the results of the ethanol-flavonoid group is not statistically significant.

Thus, the biologically active flavonoid compound (ADC) exhibits anti-SK-channel properties preventing the pathological increase in spontaneous activity of nerve cells of the central nervous system caused by exposure to ethanol. ADC effectively blocks the effect of physiological concentrations of ethanol in the blood of animals, restores coordination of movements, cognitive abilities, prevents the excitation and pathological forms of activity caused by exposure to ethanol. Therefore, the claimed biologically active flavonoid compound of the ADC is promising for the correction of behavior when intoxicated, for use as a medicine in the treatment of chronic alcohol dependence, as well as a wide range of neurological and psychiatric diseases caused by alcohol abuse.

Claims (1)

Use of the flavonoid compound 7-O- [6-O- (4-acetyl-α-L-rhamnopyranosyl) -β-D-glucopyranosido] -5-hydroxy-6-methoxy-2- (4-methoxy-phenyl) -4H -chromon-4-one (acetylpectolinarin - ADC) as a medicine for the correction of behavioral disorders: coordination of movements, attention, sober assessment of the situation, memory - caused by both short-term intoxication and chronic alcohol consumption.

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