RU2506075C1 - Sedative agent and method for preparing it - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, particularly to pharmacology and pharmaceutics, and concerns a sedative agent representing glycine immobilised on detonation nanodiamond particles 2-10 nm in size, and to a method for preparing it.

EFFECT: preparing the sedative agent.

4 cl, 7 dwg, 13 tbl, 4 ex

Description

The invention relates to medicine, in particular to pharmacology, and relates to a sedative, which is glycine immobilized on particles of detonation nanodiamonds.

Currently, psychotropic drugs that cause sedation - suppressing the reaction to constant stimuli with a decrease in the level of spontaneous activity and thinking while reducing anxiety, are widely used in medical practice, mainly for the treatment of various neurotic conditions. It is believed that such sedatives, which reduce the feeling of anxiety and have a calming effect, affect the central nervous system (CNS) without noticeable impairment of its functions, while their effect is characterized by a degree dose-dependent suppression of the central nervous system. The mechanism of action of sedatives has not yet been fully identified. Sedatives can reduce the central nervous system excitability, enhance the inhibition processes and exert a regulatory effect on the central nervous system, restoring the balance between the processes of excitation and inhibition [1].

Despite the widespread use in medicine of a wide range of sedative drugs, sufficiently effective drugs are still not available. In some cases, some drugs exhibit insufficient therapeutic effect, in others they are too “tough” and various side effects are possible with their use [1].

It is known that the replaceable amino acid glycine (NH ₂ CH ₂ COOH), being the central neurotransmitter of the inhibitory type of action, exhibits a sedative effect and improves metabolic processes in brain tissues [2]. In modern therapeutic practice, glycine is used as a means of reducing the craving for alcohol, reducing withdrawal symptoms, depressive disorders, increased irritability, and normalizes sleep, as well as in the complex treatment of cerebrovascular disorders [2]. The pharmacological action of glycine is based on the effect of amplification of metabolic and neurotransmitter processes that occur due to an increase in its endogenous synthesis. It is possible to increase the intracellular synthesis of glycine only using signal transmission pathways due to interaction with receptor systems. Its interaction with glycine receptors leads to the opening of chlorine channels, hyperpolarization of the membrane and the spread of inhibition. Along with this, glycine is able to act as an allosteric coagonist of glutamate receptors. By binding at a specific site, it enhances the ability of glutamate and N-methyl-D-asparagus (NMDA) to open the cationic channel [3,4].

The pharmacopoeial glycine used is prescribed in the form of tablets (0.1 g each) under the tongue 3-4 times a day.

Known glycine immobilized on particles of detonation nanodiamonds with a size of 2-10 nm, used as a binder component in polymer composites [5, 6]. The method of obtaining it is as follows [6]. A portion of the nanodiamond is placed in a constant helium current reactor and annealed at a temperature of 150-470 ° C for 3-4 hours. Then, the nanodiamond samples are fluorinated at a temperature of 50-500 ° C for 1-24 hours by contacting with a mixture of gaseous fluorine and hydrogen . To obtain glycine immobilized on nanodiamond particles, fluorinated nanodiamonds are sonicated in o-dichlorobenzene for 20-30 minutes, glycine ethyl ester hydrochloride (NH ₂ CH ₂ COOCH ₂ CH ₃ · HCl) and a few drops of pyridine are added. The resulting mixture was stirred at a temperature of 130-140 ° C for 8-12 hours. The resulting product was filtered, washed with ethanol and dried under vacuum at 70 ° C.

An additional characteristic of this substance is the size of its particles in suspension, equal, according to the data of dynamic light scattering (DLS), 310 nm [6].

A feature of this substance is the presence on the surface of nanodiamond particles, in addition to glycine molecules, also fluorine atoms. Although the authors declare their number to be less than 1% at., In reality it has been experimentally established that the fluorine concentration on the surface of a nanodiamond can reach 14% at. and more. This is due to the fact that the CF bond (E _St. = 115 kcal / g-atom) is strong and the fluorine derivatives of carbon are inert with respect to many substances. Therefore, during the chemical immobilization of glycine on the surface of a nanodiamond containing fluorine atoms, glycine molecules are replaced by fluorine atoms only partially. Moreover, it is known that the presence of fluorine and its derivatives in the organic matter increases its toxicity and can change the indices of the microsomal xenobiotic biotransformation system in the liver [7]. Thus, the presence of fluorine atoms in the nearest nanostructured analog of nanodiamond - fullerene (C ₆₀ ) - increases its general toxicity by 2.4–5 times [8]. In addition, fluorine and its compounds are able to accumulate in various environmental objects and be present in them in various quantities [9]. Therefore, glycine immobilized on nanodiamond particles that contain fluorine atoms is undesirable in medical practice as a medicine.

Therefore, the production of glycine immobilized on particles of detonation nanodiamonds that do not contain fluorine atoms, with a high dispersion in the suspension used as a medicine, as well as reducing the level of environmental and endoecological danger, simplifying and cheapening the method of its production, is an urgent and practically significant task .

The aim of the invention is to increase the effectiveness of the sedative effect of glycine without manifesting any side and toxic effects, reducing the dosage of the drug and expanding the range of sedative drugs.

The use of glycine immobilized on detonation nanodiamond particles as a sedative is not described in the scientific and patent literature.

The goal is achieved by using the sedative described in accordance with the invention, which is glycine immobilized on particles of detonation nanodiamonds with a size of 2-10 nm, not containing fluorine atoms and having a shell thickness of up to 1 nm, with a glycine content of up to 21 ± 3% by weight, and how to obtain it.

The described sedative in the form of glycine immobilized on particles of detonation nanodiamonds that do not contain fluorine atoms on its surface is an ultrafine powder (Figure 1) of a dark gray or dark gray color with a greenish or dark blue shade with a particle size of 2 to 10 nm, having a shell with a thickness of up to 1 nm (Figure 2), the size of the aggregates in an aqueous suspension of up to 100 nm (Figure 3) and a glycine content of up to 21 ± 3% of the mass included in the surface shell.

Figure 1 clearly shows the presence of the described sedative ultra-dispersed structure of particles with a size less than the resolution of the used device (from 20 nm).

A micrograph of the particles of the described sedative was obtained using a Zeiss Ultra Plus field emission scanning electron microscope (Carl Zeiss, Germany).

Figure 2 shows that the particle size of the described sedative, coated with a thickness of up to 1 nm, is 2-10 nm.

A micrograph of the particles of the described sedative was obtained using a Jeol 1011 transmission electron microscope (JEOL, Japan).

Figure 3 shows the curve of the distribution of particle sizes in the suspension of the described sedative, from which it follows that the particle sizes in the suspension do not exceed 100 nm.

The particle size distribution of the described sedative in suspension was measured by DLS on a ZetaSizer instrument (Malvern Instruments, USA). The abscissa represents the logarithmic scale of particle size in nm. The ordinate axis is the percentage of particles with specific sizes.

The elemental composition of the particle surface of the described sedative according to X-ray photoelectron spectroscopy (XPS) is given in Table 1.

Table 1 Elemental composition and binding energies of surface atoms of the described sedative Name of characteristic Chemical elements FROM ABOUT N Atomic% 77.5-94.5 4-14 1,5-8,5 Communication energies, eV 285.2 ± 0.5 530.7 ± 0.5 399.8 ± 0.5

The surface of the described sedative was studied using a LAS-3000 instrument (Riber, France) equipped with an OPX-150 hemispherical analyzer. To excite photoelectrons, nonmonochromatized x-ray radiation of an aluminum anode (AlK _α = 1486.6 eV) was used at a tube voltage of 12 kV and an emission current of 20 mA. Calibration of the photoelectron peaks was carried out along the C 1s carbon line with a binding energy of 285 eV. The vacuum in the working chamber was 6.7 · 10 ^-8 Pa. To obtain a high vacuum, an ion pump was used.

The amount of glycine in the described sedative is determined as follows. Mixtures of nanodiamonds with different glycine contents are prepared. Take samples of each mixture of equal weight. Their IR spectra are recorded, the most intense characteristic signals are selected on them, which are correlated with the IR spectrum bands of the initial glycine. Then build calibration curves of the dependence of the signal intensity in the IR spectrum on the glycine content in the sample. Further, by the intensity of the selected characteristic bands of the studied sedative, the quantitative glycine content in it is determined from the calibration curves. According to the data obtained, the average value of the glycine content in the described sedative is determined.

The described method of obtaining a sedative is as follows. Detonation nanodiamonds are annealed in a stream of hydrogen gas at a temperature of 500-1200 ° C for 1-8 hours, then subjected to liquid-phase chlorination with molecular chlorine by photochemical treatment with visible light at a temperature of 50-70 ° C for 36-60 hours, followed by washing with carbon tetrachloride ,

centrifugation and drying under vacuum. Chlorine-modified nanodiamonds are dissolved in a polar solvent to form a suspension. Tertiary amine and glycine are added and the resulting mixture is sonicated for 5-60 minutes, followed by exposure at 50-80 ° C for 12-48 hours, centrifugation, washing with solvent and drying. Ultrasound treatment is carried out for 5-60 minutes, triethylamine is used as a tertiary amine and pyridine, a lower aliphatic alcohol, a water-alcohol mixture or water are used as a polar solvent.

The described sedative does not contain fluorine atoms (Table 1), as well as atoms of other halogens, in an amount exceeding the error of the device (0.1% at.), Since in the process of obtaining a sedative all chlorine atoms are replaced by glycine molecules and leave from the surface of a nanodiamond in the form of HCl molecules.

In the process of pharmacological studies, work was carried out to study the specific sedative effect of the described drug and its harmlessness in comparison with pharmacopoeial glycine.

Assessment of the sedative effect was carried out according to the Guidelines for the study of the activity of substances with neuropsychotropic activity described in [10].

The specific effect was studied on white outbred male mice using a certified method - the Opto-Varimex actometer (Colambus Instrument, USA). Statistical processing of the results was carried out using BioStat software for Windows. Group averages and standard error indicators were calculated.

The results obtained indicate that pharmacopeia glycine at a dose of 10 mg / kg does not show sedative activity in the meter (Table 3-5). Also, he did not show a statistically significant sedative effect at 1, 4, and 8 hours after administration.

The described sedative caused a significant decrease in horizontal (by 67%) and vertical (by 93.6%) motor activity 10 minutes after administration. The decrease in horizontal and vertical locomotor activity remained significant and amounted to more than 50% of the control and 4 and 8 hours after a single injection of the described sedative (Table 3-5).

A study of the acute toxicity of the described sedative was carried out according to the Guidelines for the study of acute toxicity described in [10].

During the experiment, the following indicators were recorded: the nature of the coat, changes in the condition of the mucous membranes, ptosis of the upper eyelid, increased urination, increased bowel movements, increased salivation, piloeraction, vocalization, lateral position, rhythm and depth of respiratory movements, aggressiveness, timidity, tremor, convulsions, change in thresholds of pain reaction, change in posture, catalepsy, impaired coordination of movements in the test of a rotating rod, holding for 5 seconds on an inverted mesh platform, erelezanie with inverted mesh platform up, the presence of pineal, corneal reflex, sedation, stereotypes and grooming, the death of the animal.

Statistical processing of the results was carried out using statistical packages "BioStat" for Windows. Group averages and standard error indicators were calculated.

The obtained results convincingly show that the described sedative when administered intraperitoneally to mice at doses of 75, 150 and 225 mg / kg, as well as pharmacopoeial glycine did not cause signs of intoxication and death of animals over 14 days. observations.

As a result of a pharmacological study, it was established that the described sedative had a pronounced specific sedative effect superior to the sedative effect of pharmacopeia glycine, and the absence of side effects and toxic effects in a dose exceeding the therapeutic by more than 20 times.

The described sedative not only allows you to increase the therapeutic effectiveness of the pharmacopeia drug glycine, but also to expand the range of effective and safe sedatives.

A brief description of the graphic materials.

Figure 1. An electron micrograph of the described sedative obtained with a scanning electron microscope.

Figure 2. An electron micrograph of the described sedative obtained using a transmission electron microscope.

Figure 3. The particle size distribution of the described sedative in an aqueous suspension according to the DLS method.

Figure 4. IR spectra of mixtures of nanodiamonds with glycine used to construct calibration curves. I, II, III - spectra of mixtures with a glycine content of 1: 1.75: 2.5, respectively. Within the framework, characteristic peaks are highlighted.

Figure 5. Calibration curves for each characteristic band of the IR spectrum of a mixture of nanodiamonds with glycine, a, b, c - calibration curves for bands 1407, 1332 and 504 cm ^-1 , respectively.

The invention is illustrated by the following examples.

Example 1

300 mg of the initial detonation nanodiamond are annealed in a stream of hydrogen gas at a rate of 3.0 l / h at a temperature of 1000 ° C for 6 hours. Then, the annealed nanodiamond is subjected to liquid phase chlorination with molecular chlorine dissolved in 40 ml of CCl ₄ to 6 wt%. Cl ₂ . The chlorination reaction is carried out by photochemical exposure to visible light for 60 hours at a temperature of 60 ° C. The sample was then washed with CCl ₄ by centrifuging the suspension at 6000 rpm and dried under a pressure of 0.1 mm. Hg to constant weight. Then, a suspension is prepared from the chlorinated nanodiamond using 40 ml of a water-alcohol mixture (water: methanol = 1: 1), into which 300 mg of glycine as a free amino acid (NH ₂ CH ₂ COOH) is added with 1 ml of triethylamine. The resulting mixture was sonicated (50 W) for 60 minutes and kept under constant stirring at a temperature of 65 ° C for 30 hours. The resulting product was washed with a large amount of ethanol, centrifuged and dried under vacuum at 70 ° C overnight. The residual moisture content of the product is 2.2%. The yield of the target product is 279 mg (93%). The product is a dark gray with a bluish tint ultrafine powder (Figure 1) with primary particle sizes of 2-10 nm (Figure 2) having a shell of the surface layer up to 1 nm. In suspension, the particle size of the powder does not exceed 100 nm (Figure 3). The elemental composition of the particle surface of the obtained product is given in table.2.

table 2 XPS data of the obtained product Name of characteristic Chemical elements FROM ABOUT N Atomic% 80.1 ± 0.1 11.5 ± 0.1 8.4 ± 0.1 Communication energies, eV 285.2 ± 0.5 530.7 ± 0.5 399.6 ± 0.5

To determine the mass fraction of glycine in the resulting product, 3 mixtures of nanodiamond with glycine are prepared with a content of the latter 1: 1.75: 3.5, respectively. For each mixture, a weighed mass of 0.0035 g is taken and carefully ground in a mortar with 0.090 g of KBr. 0.070 g of the resulting mixture is pressed into a tablet and its IR spectrum is removed (Figure 4). Characteristic bands are selected at 1407, 1332 and 504 cm ^-1 , respectively, and calibration graphs are constructed for them (Figure 5). The intensity of the corresponding characteristic bands in the IR spectrum of the obtained sample with a mass of 0.0035 g was 0.23, 0.22, and 0.10 pu, respectively. From the calibration curves a, b, c in FIG. 5, the glycine content in the obtained sample is determined, which is 0.00057 ± 8 · 10 ^-5 g. Therefore, the mass fraction of glycine in the sample sample is 21 ± 3% of the mass.

Example 2

The study of the specific sedative activity of the described drug.

The study was performed on white mongrel sexually mature male mice weighing 25-28 g.

Animals were obtained from the Central laboratory animal nursery "Pillar", Moscow region. The keeping of animals corresponded to the rules of laboratory practice when conducting preclinical studies in the Russian Federation (GOST 3 51000.3-96 and 51000.4-96), the normative document "Sanitary rules for the design, equipment and maintenance of vivariums", approved by the Chief State Sanitary Doctor on 04/06/1973 No. 1045 -73, and Order of the Ministry of Health of the Russian Federation No. 267 of 06/19/2003 “On approval of laboratory practice rules” (GLP) in compliance with the International Recommendations of the European Convention for the Protection of Vertebrate Animals used in experimental studies (1997). The animals were kept in a vivarium at a temperature of 20-22 ° C, with a light cycle of 12 hours light and 12 hours dark periods, in T / 4 A plastic cages measuring 580 × 375 × 200 mm with a stainless steel top cover and dust-free bedding wood shavings. The animals were kept with constant access to feed and water using the full ration of extruded briquetted feed (GOST for feed P 50258-92) and drinking water. When conducting the experiments, the requirements of the Commission on the Ethics of Attitude to Animals of the Russian National Committee on Bioethics at the Russian Academy of Sciences and the ethical standards set forth in the "International Recommendations for Biomedical Research Using Animals" (1985) were taken into account. The experiments were carried out in the first half of the day.

To study the specific sedative activity of the product obtained in Example 1, a certified technique was used to evaluate various types of motor activity using an Opto-Varimex actometer (Columbus Instrument), USA), which is recommended for studying the sedative effects of substances according to the Methodological guidelines for the study of the antipsychotic activity of pharmacological substances ”[10].

As criteria characterizing sedative effect, we used indicators of horizontal and vertical locomotor activity. The movements of animals were recorded on the counter in arbitrary units. The experiment was carried out for 8 hours

The product obtained in example 1 was administered once intraperitoneally or orally (intragastrically using a special probe) at doses of 5 and 10 mg / kg. Registration of horizontal and vertical motor activity in the Opto-Varimex actometer was carried out 10 minutes, 30 minutes, 1, 4 and 8 hours after the substance was introduced. To avoid getting used to the situation and the phenomenon of extinction of motor activity, to assess the effects of the product obtained according to example 1, at each time point, separate groups of animals were used: 10 mice in each group - for control and study of the pharmacological activity of the described sedative, 6 mice in the group, on studies of the pharmacological activity of pharmacopeia glycine. A total of 88 animals were used. Pharmacopoeial glycine was used as a comparison drug. Distilled water was used as a control. Statistical processing of the results was carried out using statistical packages "BioStat" for Windows. Group averages and standard error indicators were calculated.

When studying horizontal motor activity with intraperitoneal administration, it was found that in the control group of animals, the average horizontal activity 1 hour after the introduction of distilled water was 230.96 movements. This indicator did not significantly change after 8 hours (table 3).

Table 3 The study of the sedative effect of the substance obtained according to example 1, with a single intraperitoneal administration according to the indicators of horizontal motor activity of mice in the Opto-Varimex actometer Substance Dose mg / kg Effect registration time, h The average horizontal activity (movement) in units % reduction in motor activity relative to control The control one 230.96 ± 10.23 - Sedative 10 1/6 75.78 ± 10.76 * 67.19 Sedative 10 ½ 43.46 ± 9.35 * 81.18 Sedative 10 one 21.98 ± 6.4 * 90.48 Sedative 10 four 25.49 ± 7.66 * 88.96 Sedative 10 8 74.68 ± 10.51 * 67.62 Pharmacopoeial Glycine 10 1/6 190.62 ± 18.26 17.47 Pharmacopoeial Glycine 10 one 227.08 ± 17.41 1.68 Pharmacopoeial Glycine 10 four 211.14 ± 14.70 8.59 Pharmacopoeial Glycine 10 8 219.54 ± 12.42 4.95 The control 10 8 204.57 ± 14.53 - * - p <0.01 compared with the control group, Mann-Whitney test

The described sedative obtained in example 1 at a dose of 10 mg / kg caused a significant decrease (by 67%) in horizontal motor activity 10 minutes after administration. After 30 min and after 1 h, the decrease in horizontal locomotor activity gradually increased and the decrease was 81% and 90% compared with the control, respectively (Table 3). The decrease in horizontal motor activity remained significant and amounted to more than 50% of the control and 4 and 8 hours after a single injection of the described sedative.

Pharmacopoeial glycine at a dose of 10 mg / kg with a single intraperitoneal administration did not cause a decrease in motor activity 10, 30 min, 1, 4, and 8 hours after administration (Table 3).

When studying vertical motor activity with intraperitoneal administration of substances, it was found that in the control group of animals, the average vertical activity 1 hour after the introduction of distilled water was 4.28 movements. This indicator did not significantly change even 8 hours after the introduction of distilled water (Table 4).

Table 4 The study of the sedative effect of the described drug with a single intraperitoneal administration according to the indicators of the vertical motor activity of mice in the Opto-Varimex actometer Substances Dose mg / kg Effect registration time, h The average vertical activity per minute, units % reduction in motor activity relative to control The control 10 one 4.28 ± 0.29 - Sedative 10 1/6 0.27 ± 0.09 93.67 Sedative 10 ½ 0 ± 0 * one hundred Sedative 10 one 0 ± 0 * one hundred Sedative 10 four 0.01 ± 0.01 * 99.77 Sedative 10 8 0.37 ± 0.08 * 91.36 Pharmacopoeial Glycine 10 one 3.34 ± 0.35 ^# 21.73 Pharmacopoeial Glycine 10 four 2.38 ± 1.32 * 44.39 The control 10 8 3.73 ± 0.35 - * - p <0.01 compared with the control group, Mann-Whitney criteria # - p <0.05 compared with the control group, Mann-Whitney test

The described sedative at a dose of 10 mg / kg with a single intraperitoneal administration caused a pronounced significant decrease (by 93.6%) in vertical motor activity 10 minutes after administration. After 30 minutes and 1 hour after a single injection, it completely eliminated vertical motor activity. A significant decrease in vertical locomotor activity persisted after 4 hours (by 99%) and after 8 hours (by 89.9%) (Table 4).

Pharmacopoeial glycine at a dose of 10 mg / kg with a single intraperitoneal injection, both after 1 h and 4 h after administration, statistically significantly reduced the vertical motor activity compared to the control (Table 4).

Example 3

The study of the specific sedative activity of the described drug with oral (intragastric) administration.

The study was performed on white outbred adult mice males weighing 25-28 g. The experimental animals were kept as in Example 1. At each time point, separate groups of animals were used: 10 mice in each group — for control and study of the pharmacological activity of the described sedative, 6 mice in the group - to study the pharmacological activity of pharmacopoeial glycine. A total of 62 animals were used.

When studying horizontal motor activity during oral (intragastric) administration of substances, it was found that in the control group of animals, the average vertical activity 30 minutes after the introduction of distilled water was 208.4 movements (Table 5).

Table 5 The study of the sedative effect of the described drug with a single oral (intragastric) administration according to the indicators of horizontal motor activity of mice in the Opto-Varimex actometer Substance Dose mg / kg Effect registration time, h The average horizontal activity (movement) per minute, units % reduction in motor activity relative to control The control ½ 208.40 ± 21.52 - Sedative 10 ½ 110.03 ± 19.94 * 47.21 Sedative 10 one 67.76 ± 11.12 * 67.51 Sedative 10 four 83.67 ± 9.81 * 59.85 Sedative 10 8 94.34 ± 7.56 * 54.73 Pharmacopoeial Glycine 10 one 189.24 ± 23.13 17.47 Pharmacopoeial Glycine 10 four 201.75 ± 21.64 3.21 * - p <0.05 compared with the control group, Mann-Whitney test

It was found that already 30 minutes after the administration of the (oral) described sedative at a dose of 10 mg / kg, a statistically significant decrease in the motor activity of animals was observed by a factor of 1.9 compared with the control group. After 1 hour after administration, a statistically significant decrease in the motor activity of animals was maintained and the level of activity was also 1.9 times lower than in the control group. After 4 hours, motor activity remained statistically significantly reduced at the same level. After 8 hours, a statistically significant decrease in the motor activity of animals was observed by 1.5 times in comparison with the control group (Table 5). The results obtained indicate that a single oral (intragastric) administration of the described sedative at a dose of 10 mg / kg causes a persistent statistically significant sedative effect within 8 hours of observation.

Pharmacopoeial glycine at a dose of 10 mg / kg after a single oral administration (intragastric) both 1 hour and 4 hours after the administration statistically significantly reduced the vertical motor activity compared to the control (Table 5).

Example 4

The study of side effects and toxic effects of the described sedative

The study was conducted on white mongrel sexually mature male mice weighing 20-24 g, 2-3 months old. The experimental animals were kept as in Example 2. A total of 42 animals were used — 7 groups of 6 mice each.

Changes in behavior, reflexes, possible side effects, signs of intoxication, and death of animals were recorded after 1, 4, 24 hours, 4, 10, and 14 days. after intraperitoneal administration of the described sedative in comparison with the pharmacopeia substance of glycine in the same doses.

As a result of the study, it was found that the described sedative when administered intraperitoneally to mice at doses of 75, 150 and 225 mg / kg did not cause signs of intoxication and death of animals over 14 days. (tab. 6-11). At the same time, the described sedative also did not cause changes in the coat of the mice, the state of the mucous membranes. There was also no ptosis of the upper eyelid, increased urination, defecation, salivation, piloerection, vocalization, lateral position. Within the normal range, there was a rhythm and depth of respiratory movements, there was no aggressiveness, timidity, tremor, convulsions, catalepsy, stereotype and grooming. No change in posture was observed. The animals retained pineal, corneal and pain reflexes. For all 14 days. observations, the animals were kept on an inverted mesh platform for 5 s (Table 6-11).

When studying the reaction to pain irritation by squeezing the base of the tail of the mouse with tweezers, it was found that after administration of the described sedative in animals, a reduced response to the pain stimulus was observed in the first 4 hours of observation, as compared to the control, in terms of head-to-tail rotation and squeak. After 24 hours of observation, normalization of the reaction of the animal to a pain stimulus was noted. Pharmacopoeial glycine at a dose of 75 mg / kg did not cause a change in the response to the pain stimulus for all 14 days. after administration.

The study of the effect of the described sedative in comparison with pharmacopoeial glycine on motor activity and coordination of movements was carried out using a rotating rod test (Rota Rod company Ugo Basile, Italy). The fixed rod rotation speed was 10 rpm. The criterion for completing the task was to keep the animal on a rotating rod for 120 s. Impaired coordination of movements was observed an hour after the administration of the described sedative at a dose of 75 mg / kg in 16.7% of animals and in 50% when the drug was administered in doses of 150 and 225 mg / kg After 24 hours, 4, 10 and 14 days. violations of motor activity under the influence of the described sedative at all doses were not observed (tab. 6-12).

Table 6 The study of possible side effects and death of mice 1 hour after the administration of pharmacopoeial glycine and the sedative described (indicator of change in the group in%) Indicators Groups The control Sedative / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 The presence of pilo erection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 33.3 * 0 0 33.3 * Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 66.7 * one hundred* one hundred* 0 one hundred* one hundred* Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 16.7 fifty* fifty* 33.3 * 33.3 * fifty* 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred 66.7 * 66.7 * fifty* 66.7 * 66.7 * 66.7 * The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 66.7 * one hundred* one hundred* 0 one hundred* one hundred* The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 7 Study of possible side effects and death of mice 4 hours after the administration of pharmacopoeial glycine and the sedative described (indicator of change in the group in%) Indicators Groups The control Sedative / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 The presence of pilo erection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 16.7 0 0 16.7 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 66.7 * one hundred* one hundred* 0 one hundred* one hundred* Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 16.7 16.7 fifty* 33.3 * 16.7 33.3 * 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred 66.7 * 66.7 * fifty* one hundred* 83.3 66.7 * The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 66.7 * 83.3 * one hundred* 0 83.3 * one hundred* The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 8 Study of possible side effects and death of mice 24 hours after the administration of pharmacopeia glycine and the sedative described (indicator of change in the group in%) Indicators Groups The control Sedative / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 The presence of pilo erection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change pain thresholds 0 0 0 0 0 0 0 reactions Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 33.3 * 16.7 33.3 * 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred 66.6 * The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 0 0 0 0 0 0 The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 9 Study of possible side effects and death of mice after 4 days. after the administration of pharmacopeia glycine and the described sedative (indicator of change in the group in%) Indicators Groups Comte
role Sedative / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 The presence of pilo erection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 0 0 0 0 0 0 Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 0 0 0 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 0 0 0 0 0 0 The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 10 The study of possible side effects and death of mice after 10 days. after the administration of pharmacopeia glycine and the described sedative (indicator of change in the group in%) Indicators Groups Comte
role Sedative / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 The presence of pilo erection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 0 0 0 0 0 0 Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 0 0 0 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred 83.3 The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 0 0 0 0 0 0 The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 11 The study of possible side effects and death of mice after 14 days. after the administration of pharmacopeia glycine and the described sedative (indicator of change in the group in%) Indicators Groups Comte
role Sedative / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 The presence of pilo erection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 0 0 0 0 0 0 Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 0 0 0 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred 66.7 * The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 0 0 0 0 0 0 The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 12 The effect of the described sedative in comparison with pharmacopoeial glycine on the motor activity of mice in the test of the rotating rod Group Dose mg / kg Group indicator,% 1 hour 4 h 24 h 4 days 10 days 14 days The control - 0 0 0 0 0 0 75 16.67 16.67 0 0 0 0 Sedative 150 fifty* 16.67 0 0 0 0 225 fifty* fifty* 0 0 0 0 75 33.33 * 33.33 * 33.33 * 0 0 0 Pharmacopoeial Glycine 150 33.33 * 16.67 16.67 0 0 0 225 fifty* 33.33 * 33.33 * 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Pharmacopoeial glycine caused impaired coordination of movements at doses of 75, 150 mg / kg in 16.7% and 33% of animals during 24 hours of observation, and at a dose of 225 mg / kg in 50% of mice 1 hour after administration and in 33% 4 and 24 hours after administration.

Thus, impaired coordination of movements after the introduction of the described sedative was recorded mainly during the first 4 hours of observation. Subsequent observations (24 hours, 4, 10 and 14 days) showed no differences between the study group and the control. Violation of coordination of movements after administration of pharmacopoeial glycine to animals persisted 24 hours after administration.

When studying the muscle relaxant effect of the described sedative and pharmacopeia glycine in the test of keeping mice on an inverted mesh platform for 5 sec, no differences with respect to the control were revealed within 14 days. observations.

When studying the motor activity of mice, it was found that both pharmacopeia glycine and the sedative described described impaired the ability of animals to climb onto the top of an inverted mesh platform. So, in the first hour of observation in all experimental groups there was a decrease in the number of animals capable of completing the task. On the 4th, 10th and 14th day. after the introduction of substances, there was no violation of the ability to move to the top of the mesh platform.

To study the sedative effect on the ability to move on a plane, the animal was placed on a laboratory table. The absence of movements in the animal during the first 5 s was regarded as the presence of a sedative effect. The results of the study are presented in table 6-11 and table 13. It was found that with the introduction of the described sedative (75, 150 and 225 mg / kg) and pharmacopeia glycine (150 and 225 mg / kg), pronounced sedation was observed in the first 4 hours, which had a dose-dependent character. Sedation during the first 4 hours was absent only in the group of animals treated with pharmacopoeial glycine at a dose of 75 mg / kg. After 1.4, 10 and 14 days. after the introduction of substances sedation was not recorded. These data confirm the presence of the described sedative in a more pronounced sedative effect in the test of motor activity of mice compared with pharmacopoeial glycine.

Table 13 The study of the sedative effect of the described drug in comparison with pharmacopoeial glycine in mice in the test of rapping on the cell Group Dose mg / kg Group indicator,% 1 hour 4 h 24 h 4 days 10 days 14 days The control - 0 0 0 0 0 0 75 66.67 * 66.67 * 0 0 0 0 Sedative 150 one hundred* 83.33 * 0 0 0 0 225 one hundred* one hundred* 0 0 0 0 Pharmacopoeial Glycine 75 0 0 0 0 0 0 150 one hundred* 83.33 * 0 0 0 0 225 one hundred* one hundred* 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

In the test of tapping on the animal’s cell, it was found that in the group in which the described sedative was administered to the mice at a dose of 225 mg / kg, during the first 4 hours there was no reaction of the mice to pencil tapping on the animal’s cell compared to the control group, which can be considered as an expression of the sedative effect of the described drug. Subsequent observations after 1.4, 10 and 14 days. there were no differences between the groups (Table 6-11).

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Claims (4)

1. Sedative, which is glycine immobilized on particles of detonation nanodiamonds with a size of 2-10 nm, having a shell thickness of up to 1 nm, with a glycine content of up to (21 ± 3) wt.%. 2. The method of producing a sedative according to claim 1, characterized in that the detonation nanodiamond is annealed in a stream of hydrogen gas at a temperature of 500-1200 ° C for 1-8 hours, subjected to liquid-phase chlorination with molecular chlorine under photochemical exposure to visible light at a temperature of 50- 70 ° C for 36-60 hours, followed by washing with carbon tetrachloride, centrifugation and drying, the resulting chlorine-modified nanodiamond is dissolved in a polar solvent to form a suspension, tertiary amine and glycine are added The resulting mixture was sonicated followed by standing at 50-80 ° C, centrifugation, washing with solvent and drying. 3. The method according to claim 2, where the ultrasound treatment is carried out for 5-60 minutes and keeping at 50-80 ° C is carried out for 12 to 48 hours 4. The method according to claim 2 or 3, where triethylamine is used as a tertiary amine and pyridine, a lower aliphatic alcohol, a water-alcohol mixture or water are used as a polar solvent.

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