RU2155065C1 - Anxiolytic agent and pharmaceutical composition with anxiolytic effect - Google Patents

Abstract

FIELD: medicine, peptides. SUBSTANCE: invention relates to development and use of the novel anxiolytic agent. Invention proposes to use heptapeptide Thr-Lys-Pro-Arg-Pro-Gly-Pro as an anxiolytic agent and the novel composition of anxiolytic agent containing indicated heptapeptide as an active substance in amount of 1.45-1.55 g/l, nipagine as a preserving agent in amount of 0.95-1.05 g/l and distilled water, the balance. Composition is used as nasal drops. EFFECT: high effectiveness in low doses, simplified using. 2 cl, 19 tbl, 14 dwg, 11 ex

Description

 The invention relates to medicine, namely to the development and use of a new anxiolytic agent. It can be used for treatment aimed at increasing motivational stability and adequate adaptive behavior in psychiatric and neurological diseases to correct fear, anxiety, which form the pathogenetic basis of many psychoemotional disorders. For the treatment and rehabilitation of patients with neurosurgical operations and craniocerebral injuries, as well as to increase the adaptive and adaptive capabilities of a healthy person in extreme situations.

 The relevance of the development of the drug is determined by the increase in stressful situations in modern life associated with technological and natural disasters, the deteriorating environmental situation, urbanization and the instability of the political and economic situation in modern society, the general tendency to aging society and the lack of anxiolytics in modern medicine that do not have side effects.

 Various tranquilizers, mainly of the benzodiazepine series, which are widely used in clinical practice, are known as anxiolytics. However, they are characterized by a large number of side effects: sedation, muscle relaxation, hypo-sedative, hypnotic effect, impaired learning, the phenomenon of withdrawal and others.

 A new direction in the field of creating effective and safe medicines for adequate relief of fear and anxiety is the creation of anxiolytics based on endogenous regulatory peptides that are highly effective and harmless due to their belonging to biological structures related to the body.

 Known heptapeptide of the General formula (Thr-Lys-Pro-Arg-Pro-Gly-Pro) as a memory stimulant (USSR Patent N 1124544, priority June 30, 1983). However, the possibility of using it as a medication of anxiolytic action is unknown. The technical result achieved by the implementation of the invention is the discovery of a wide range of therapeutic effects of the known memory stimulant, which determines the possibility of its use in low doses as an anxiolytic agent, in the form of a convenient dosage form without undesirable side effects characteristic of tranquilizers, with good tolerance .

 The severity of the anxiolytic effect of this new drug in doses of 200-300 mcg / kg is more comparable with the effects of hydazepam (7 mg / kg), diazepam (0.3 mg / kg), phenazepam (0.1 mg / kg), alprazolam (0.2 mg / kg) - any of which can be considered as a prototype of the proposed new drug. However, unlike the above tranquilizers, which are considered as a prototype, an increase in the dose of a new drug by 30-50 times and up to 200-500 times in various tests is not accompanied by the development of undesirable side effects.

 The above technical result is achieved by the fact that the known heptapeptide of the general formula: Thr-Lys-Pro-Arg-Pro-Gly-Pro is used as an anxiolytic agent.

In the medicinal composition of the anxiolytic agent containing the active substance and distilled water, the heptapeptide Thr-Lys-Pro-Arg-Pro-Gly-Pro and the additional preservative nipagin are contained as the active substance in the following ratio of components, g / l:
Heptapeptide Thr-Lys-Pro-Arg-Pro-Gly-Pro - 1.45-1.55
Nipagin - 0.95-1.05
Distilled Water - Else
The dosage form is named Selank.

 Further in the text, the dosage form will be called Selank, and the active substance heptapeptide Thr-Lys-Pro-Arg-Pro-Gly-Pro is simply a heptapeptide.

 It was found that the heptapeptide Thr-Lys-Pro-Arg-Pro-Gly-Pro, known as a memory stimulant, has a wide spectrum of action. Along with the anxiolytic effect, the heptapeptide has an optimizing and activating effect on the mnestic and cognitive functions of the brain, which is an important positive part of its specific pharmacological activity. Heptapeptide activates the processes of learning and memory (fixation, consolidation, reproduction in normal animals and in the experimental pathology of these brain functions). These properties distinguish the pharmacological action of the heptapeptide from the pharmacological action of tranquilizers.

List of abbreviations:
VMG - ventromedial nucleus of the hypothalamus
OP - open field
OR - indicative reactions
PKL - an elevated cruciform labyrinth
TSK - dark light camera
Passive avoidance reaction - conditional reaction of passive avoidance
URAI - a conditional reaction of active avoidance
ESD - emotional stress response
Example 1. The effect on the behavior of rats in the test of the conflict situation caused by the collision of the defense and food reflexes.

 The method of a conflict situation based on the collision of drinking and defensive reflexes under the action of a sudden pain or aversive stimulus is generally accepted for detecting the tranquilizing and anxiolytic effects of compounds (T.A. Voronina et al, 1982). Depending on the strength of the current, the animal can receive intense pain ("damaging") irritation or aversive ("punishing", rejected), emotionally negative.

 The experiment began with the development in rats of the skill of taking water from a drinking bowl installed in the experimental chamber. 48 hours before the experiment, the animals were deprived of water and fed only with dry food (drinking deprivation). Then, every day (for 3 days) at the same time of the day, the rats were placed in an experimental chamber (for 10 minutes), where they could take water from the drinker. On the 4th day of training (after stabilization of the drinking reflex), the rats were again placed in the experimental chamber, but five seconds after the start of drinking, electrical irritation was applied by passing current through the drinker. The current intensity of two intensities was used: 1 mA - suprathreshold pain irritation; 0.25 and 0.35 mA - threshold pain or aversive, emotionally negative. The collision of the two reflexes, drinking and defensive, created an extreme conflict situation in which the fear of receiving pain (damaging) or aversive (negative, rejected) irritation kept animals from taking water, despite drinking deprivation. Within 20 minutes, the following were recorded: the amount of water taken from the drinker, despite receiving electrical irritation; the number of approaches to the drinker; general motor activity and general emotional state according to such signs as: voice reactions, inadequate response to the presented test stimuli, sudden fluctuations in breathing, visible tachycardia. The heptapeptide was administered intranasally at doses of 100 to 500 μg / kg. To compare the effectiveness of the heptapeptide with a drug that has an anxiolytic effect under these experimental conditions, a benzaziazepine tranquilizer diazepam (at a dose of 1 mg / kg) was used intraperitoneally.

 It was found that heptapeptide in doses from 100 to 500 μg / kg significantly increases the number of water taken from the drinking bowl when using aversive stimulation (current strength I = 0.25 mA and 0.35 mA). The most effective doses were 250 μg / kg and 300 μg / kg, at which the number of water intake from the drinker increased by an average of 2.3 times, the number of approaches without water intake increased by 1.5 times (P <0.05) (Table 1).

 Many animals did not leave the drinking bowl and often made movements resembling unfinished attempts to take water. The externally visible manifestations of emotional stress (fear) observed in the control were replaced by research activity with a return to the drinker. When using a current of 1 mA (intense pain irritation), the amount of water taken from the drinker also increased, however, it did not reach the reliability criterion, however, the animals more often approached the shelf, visually recorded signs of anxiety-anxiety (pilo erection, voice reactions, muscle tension ) were eliminated. In animals treated with diazepam (1 mg / kg), a highly significant (P <0.01) increase in the number of attempts to take water (Fig. 1) was observed both with the use of aversive and intense pain irritation. Despite repeated electric shocks, the animals not only approached, but also took water from the drinking bowl.

 Thus, the results indicate that, in the conflict model, the heptapeptide has an effect similar to the classical diazepam tranquilizer and associated with eliminating the fear of receiving an aversive effect. However, with a 4-fold increase in the intensity of the current strength, the effect of the heptapeptide weakens, while diazepam persists. This observation apparently reflects the fact that the adequacy of the assessment of the situation against the background of the action of the heptapeptide is preserved, despite the decrease in the negativeness of its perception.

 Example 2. The effect on the behavior of mice with a hereditarily controlled phenotype of emotional stress response in the tests of the elevated cruciform labyrinth (PCL).

 The elevated cruciform labyrinth (PCL) method is currently used in world and domestic experimental psychopharmacology as an adequate test for detecting the anxiolytic properties of known drugs and new compounds (Pellow Sh. Et. Al., 1985; Yu.A. Blednov et al. 1994; SB Seredenin et. Al 1995). A negative situation is created by the collision of two opposite natural forms of behavior of different modalities for rodents: anxiety and search activity. It is important that the experiment is conducted without the use of harmful (noxions) or damaging stimuli, such as electroshock, pain irritation, manipulations with food deprivation, etc. The experiments were performed on mice with a “passive” ESR phenotype and a high level of anxiety (Balb / c line) and the "active" ESR phenotype (line C57BL / 6) (S.B.Seredenin, Vedernikov, 1979; S.B.Seredenin et al., 1989; Yu.A. Blednov et al., 1994). According to the standard program, the following were recorded in the experiments: time spent in the “open” (light) and “closed” (dark) arms of the PKL, total time spent in the light compartments, in the dark arms, the total number of movements and the ratio of the number of runs in the light arms to the total number races, observation duration - 5 minutes. The heptapeptide was administered at doses from 100 to 6000 μg / kg ip 30 minutes before testing. Statistical processing is given using the method of one-way analysis of variance followed by intergroup comparison according to the Newman-Cueps method (Fig. 2 and 3).

 It was found that the heptapeptide in the entire range of tested doses from 100 to 3000 μg / kg dose-dependently increases the residence time of Balb / c mice in the light arms and the center of the PKL (Fig. 2 A, B). The number of runs in the light sleeves of the PKL increases by almost 3 times.

 It can be seen that diazepam in doses of 0.5-1.0 mg / kg had a similar effect on the behavior of Balb / c mice in the PKL test with heptapeptide, i.e. increasing the time spent in the open arms of the labyrinth, increasing (albeit less significantly) the motor activity and the percentage of runs in the open arms (Table 2); however, at a dose of 2 mg / kg, it had, unlike the heptapeptide, a significantly significant sedative effect: overall motor activity decreased sharply, the delay time in the center of the maze increased. Diazepam had a sedative effect on the behavior of C57BL / 6 mice in lower doses of 0.5-1.0 mg / kg (Table 2).

 Thus, in the PKL test, the heptapeptide, like diazepam, had an anxiolytic effect on the behavior of Balb / c mice, the heptapeptide had practically no effect on the behavior of C57BL / 6 mice in the PKL test, while diazepam had a sedative effect on mice of this line already at a dose of 0.5 mg / kg The anxiolytic effect of the heptapeptide on the behavior of Balb / c mice was not accompanied by a decrease in motor activity even with a 30-fold increase in dose, while a 2-fold increase in the dose of diazepam caused a decrease in motor activity. The domed dose-response relationship observed with diazepam administration reflects, according to a number of authors (Yu.A. Blednov et al., 1995), the development of a common sedative and muscle relaxant action typical of benzdiazepines. The absence of this kind of dependence in the action of the heptapeptide characterizes it as an anxiolytic, devoid of the side (sedative, muscle relaxant) effects of benzodiazepine tranquilizers.

 Thus, the use of a pharmacogenetic methodological approach and a highly specific method for detecting the anxiolytic effect of drugs of the PKL test made it possible to establish the presence and selectivity (with respect to side effects of benzodiazepines) of the anxiolytic effect of the studied drug. In a wide range of doses (from 100 to 3000 μg / kg) without any side effects, the anxiolytic effect of the heptapeptide in the PCL test is formed in mice with an initially passive form of ESD and a high level of anxiety. In the same dose range, the heptapeptide practically does not change the behavior of mice with an active form of ESD and initially low anxiety.

 Example 3. The influence of the open field (OP) and dark-light chamber (TSC) on the reaction of hiding in tests (preference reaction).

 It is known that the basis of a psychoemotional reaction to stress is an emotionally negative state, fear and anxiety. The nature and strength of the emotional stress response is hereditarily controlled. The experiments were performed on Balb / c and C57BL / 6 mice with the opposite hereditarily controlled “passive” and “active” ESR phenotype. Two standard models for reproducing the stress response in mice were used: unavoidable stress according to the Grawby I.N. method. using the installation of the "dark light camera" (TSC) and the open field test (OP).

 The following were recorded in the experiments: the number of crossed squares on the periphery and in the central regions, the number of vertical posts, the total-total number of movements (total motor activity). Current and previous studies have shown that the behavior of mice of the Balb / c (c) and C57BL / 6 (B6) lines in the stress situation of OP is fundamentally different. In line C mice, a freezing reaction occurs. Hiding at the wall of the labyrinth, very low locomotor activity within 1-3 squares in the peripheral part of the OP, there are practically no racks, there are no exits to the central parts of the OP. In mice of the B6 strain, on the contrary, activity was more than 6 times higher; frequent stays and exits to the central parts of the OP were observed.

 The heptapeptide was administered intraperitoneally at doses of 200 to 1000 μg / kg 30 minutes before testing. It was found that in the entire range of tested doses, the heptapeptide exerts a pronounced antistress effect only in line C mice with the “passive” ESR phenotype (Tables 3, 4). In conditions of OP, the peptide eliminates environmentally significant manifestations of anxiety anxiety. Freezing reaction does not occur, motor activity increases by 2 times, horizontal movements occur both in the peripheral and central zones, and the animal re-crosses the center of the field. The number of uprights is increasing (Table 3).

 In the same dose range, the heptapeptide does not significantly affect the behavior of mice with the initially "active" ESR phenotype (Table 4).

 The heptapeptide also did not exert a significant effect on the spontaneous locomotor activity of line C mice located in the "home cage" outside of a conflicting stressful situation (Table 6). And it did not cause sedation, depression, muscle relaxation, accompanying, as you know, the anxiolytic effect of known tranquilizers on the behavior of mice and rats in the test OP.

 The effect of the heptapeptide on the behavior of mice of both strains in the dark-light chamber test (preference reaction or avoidable stress) was studied. The reaction was modeled by the method of Grawby I.N. using the installation "dark light camera" (TSC). The heptapeptide was administered in the same manner at doses of 200 and 400 μg / kg. The following were recorded: total motor activity according to the number of crossed squares, the number of entries into the dark chamber, the latent period of the first entry, and the total time spent in the light or dark parts of the chamber. The duration of observation is 5 minutes. The initial behavior of c and B6 mice in this stress situation was fundamentally different. In mice, initially disturbing (line c), the number of crossed squares was 5 times less, the latent period of entry into the dark compartment was 8–9 times shorter than in mice of the B6 line. Having discovered the possibility of avoiding a stressful situation by entering a dark chamber, the mice no longer left it. The time spent in the dark chamber reached 87% of the total observation time. In contrast, the B6 mice examined the territory for a long time without going into the dark part of the TSC installation; the time spent in the dark chamber was 17% of the total time.

 It was found that the heptapeptide fundamentally changed the behavior of the highly anxious animals of the c line in this stressful situation. The time spent in the dark chamber was reduced by 7 times, and most of the animals did not leave the light chamber at all, continuing to explore the brightly lit area. The latent period of the first approach increased by almost 30 times. No significant activating effect was detected in B6 mice (Table 5).

 Thus, in mice with a highly anxious genetically determined “passive” type of emotional-stress response (Balb / c line), the anxiolytic effect of the heptapeptide in open-field (OP) and dark-light chamber (TSC) situations was revealed. In mice with an "active" form of ESD (line C57BL / 6), the anxiolytic effect of the heptapeptide on the same models and in the same dose ranges is not detected. Unlike the action of diazepam, the anxiolytic effect of the heptapeptide develops without side effects.

 In order to further analyze the selectivity of the anxiolytic effect of the heptapeptide, which in Balb / c mice is accompanied by "activation" of motor activity in the open arms of the PKL, elimination of the freezing reaction and increased motor activity in the tests of OP and TSC, a special series of experiments was conducted to identify the effect of the drug on spontaneous motor activity. The experiments were performed without moving the mice to an unfamiliar situation, i.e. a group of 10 animals located in a familiar ("home") cage was mounted on a special platform (supported by piezoelectric sensors), also equipped with photocells recording the movements of mice fixed on the recorder. As can be seen from the table. 6, the heptapeptide in a wide range of doses (from 200 to 1200 μg / kg) did not significantly affect the spontaneous locomotor activity of animals, when they were at rest in the "home" cage.

 Thus, the effect of the heptapeptide is not the result of a direct activating effect on locomotor activity, since out of a stressful situation spontaneous locomotor activity does not significantly change with the same doses. The data reflect the selectivity of the anxiolytic action of the heptapeptide, since the elimination of anxiety-fear and "activation of behavior" in a stressful situation is detected only on Balb / c mice (highly anxious, with a "passive" ESR phenotype). In mice of both strains, there was also no significant decrease in motor activity even against the background of very large doses of the drug.

 Example 4. The effect on the deliverance reaction of rats of different topological affiliation in the Henderson diving test.

 To study the effect of the heptapeptide on the emotional-stress response of rats and the ability to realize stress relief, the method proposed by Hendtrson (1970) in the modification of N.A. Bondarenko (1980). The animals were placed in a glass cylinder with a diameter of 12 cm and a height of 22 cm. The lower end of the cylinder was immersed in a "pool" of water (pool depth - 50 cm) to a depth of 2.5 cm. A ladder was placed on the inside of the pool. An animal immersed in a cylinder filled with water (tail down) could, having overcome fear, plunge headlong into the water and, diving under the walls of the cylinder, find a ladder and climb to the edge of the pool. Such a complex way of getting rid of requires an animal decision that is modulated by its emotional state. The animal was placed in a cylinder for 2 minutes. The following were recorded: the latent period of the first attempt to rid, the number of unsuccessful attempts to rid (error), the number of indicative movements in the cylinder, the level of affective manifestations (vocalization). The heptapeptide was administered intraperitoneally at doses from 70 to 1200 μg / kg and, in a series of experiments, intranasally and intraperitoneally at the most effective dose - 300 μg / kg.

 It was found that the heptapeptide in the dose range from 100 to 1200 μg / kg had a “calming” (tranquilizing) effect on the behavior of highly emotional animals of the first group: the severity of affective reactions decreased, the number of erroneous actions significantly decreased, almost all animals quickly realized the avoidance reaction, diving under cylinder walls (table. 7).

 There is a pronounced dose-dependent effect with an optimal expression at a dose of 300 mcg / kg. Against the background of a large dose (1200 μg / kg), the total number of movements of the animal in the cylinder decreases, the latent period of the release reaction increases, but the reaction itself is carried out clearly. In animals of the second group, with a low level of ESD, in this situation the latent period of disposal is slightly increased, while increasing the dose to 1200 μg / kg does not have an additional effect on behavior. In a separate series of experiments, the effect of heptapeptide at the most effective dose (300 μg / kg) was compared with intraperitoneal and intranasal methods of administration (Table 8).

 It was found that differences in the action of heptapeptide with different methods of its administration to rats are not detected, which confirms the similar results obtained in other experimental models.

 Thus, the heptapeptide exerted a pronounced antistress effect and contributed to the implementation of the behavior of ridding rats from an acute stress situation. Moreover, the dynamics of the components of stress-induced behavior against the background of the heptapeptide depended on the characteristics of the individual typological response of animals to a stress situation.

 The data indicate the modulating effect of the heptapeptide on emotional reactivity and its ability to optimize the behavior of getting rid of stress situations.

 Example 5. The effect on the anxiety-fear reaction in the test of the escape reaction during electrical stimulation of the "trigger zones of fear" of the ventromedial nucleus of the hypothalamus (VMG) of rabbits.

 The method of direct electrical stimulation of genetically determined motivational centers of the hypothalamus is traditionally used in experimental psychopharmacology (A.V. Waldman, M.M. Kozlovskaya, 1969; A.V. Waldman and coauthors, 1976; 1979; K.V. Sudakov, 1971, 1996 ) With electrical irritation of the “centers of fear” of the ventromedial hypothalamus (KV Sudakov, 1996), rabbits develop defensive behavior, fading, jumping, running away and a long-lasting state of anxiety.

 In our experiments, the anxiolytic, anti-anxiety effect of the heptapeptide was studied on male Chinchilla rabbits (weighing 3000 - 3200 g) with bipolar electrodes stereotoxically implanted into the ventromedial nucleus of the hypothalamus according to the previously published method (V.I. Badikov et al., 1985). Parameters of the testing stimuli: the frequency and duration of rectangular pulses - 100 Hz and 0.3 Msec, the duration of repeated (every 10 minutes) stimulations of 1-4 seconds (depending on the reaction of the animal), with a total experiment duration of 3 hours. The testing amplitude of stimulation (amperage) was selected individually until the reaction "jump-shoot" and remained constant throughout the experiment. In the 1-4-second interval of direct stimulation of VMH in the rabbit, there was instant alarming of the "alarm" type, jump and escape (escape reaction), limited by the size of the experimental chamber. At 10-minute intervals between irritations, the reaction of emotional stress, anxiety, and the transformation of the general structure of behavior typical of an animal persisted.

 The following were recorded in the experiments: the latent period of the escape reaction and changes in the structure of behavior, reflecting the state of emotional stress and anxiety. Every 10 minutes of the interstimulus period, the duration (in seconds) of different phases (patterns) of behavior that are part of the general structure of spontaneous behavior of rabbits was measured. The following phases were taken into account: emotional stress (a characteristic motionless posture on straightened paws with ears raised high, muscle tension, sharp jerking and escaping to afferent stimuli), orientational reaction (RR), aggressiveness (spontaneous or provoked knocking on feet, biting objects), grooming, food (approach to the feeding trough, food), drinking (approach to drinking, drinking) reactions, the state of "rest" or "comfort" (quiet inaction on the verge of falling asleep, muscle relaxation, covering the eyes). In each animal, on the 1st day of observation, the typical structure, turnover and duration of the divided six phases of spontaneous behavior were recorded, on the second day - the dynamics of the change in the structure of behavior against the background of anxiety caused by stimulation, on the third day - the same after the preliminary introduction of heptapeptide or distilled water (control).

 The heptapeptide was administered at a dose of 200 mcg / kg intranasally 15 minutes before testing. Mathematical processing of the results was carried out individually for each animal and with an assessment of the effect of the drug in the group of animals (10 rabbits). The absolute (in sec) and relative (in%) content of each phase of behavior in each 10-minute time interval, as well as in total within the first, second, and third hours of observation, was taken into account. It was found that the electrical stimulation of VMH (“centers of fear”), causing an escape reaction with a latent period of 1-2 seconds, significantly changes the behavior structure of rabbits (Table 9).

 The dominant (P <0.05) phase is the phase of emotional stress, which is a component of emotional stress and anxiety. The total duration of the phase of emotional stress increases by almost 40 times, while behavioral patterns associated with a state of rest, comfort, or the satisfaction of one's own motivations (eating, drinking) are suppressed, their duration is reduced by more than 5 times (total). Against the background of the heptapeptide, the duration of the phase of emotional stress is reduced by 2.5 times (P <0.05), while the duration of the comfort phase, on the contrary, increases by 2.3 times (P <0.05). The duration of the phases of eating and drinking behavior is increasing. The data presented in table. 9, in general, illustrate the positive dynamics of the restoration of the structure of spontaneous behavior of rabbits (n = 10) against the background of the heptapeptide and a significant decrease in the level of emotional stress in the 3-hour interstimulus period. With direct stimulation of VMH ("centers of fear") against the background of the heptapeptide, it is not completely suppressed, however, the latent period of its realization increases (Fig. 4). Thus, the anxiolytic effect of the heptapeptide was detected with different methods of administration (intravenously, intraperitoneally, intranasally) using conventional tests. The anxiolytic effect is comparable to the action of known tranquilizers (phenazepam, alprazolam, hydazepam). The data revealed the selectivity of anxiolytic action and the absence of side effects (muscle relaxation, sedation, drowsiness), characteristic of benzdizepines. Thus, a heptapeptide in a wide range of doses (from 100 to 6000 μg / kg) exerts a selective (selective) anxiolytic effect.

 Studies have shown that the heptapeptide not only does not cause side effects characteristic of tranquilizers (muscle relaxation, sedation, drowsiness), but also has a pronounced positive effect on mnestic functions, which is confirmed by the following examples.

 Example 6. The effect on the conditioned reflex of passive avoidance.

 The development of a conditioned passive avoidance reflex (passive avoidance reaction) was carried out according to a modified Buresh method. The experimental setup consisted of 2 chambers of equal size (25x25) with an electrode floor separated by a partition with a small hole for the rat to move from one chamber to another.

 The mouse was placed in the light chamber with its tail to the viral opening and the residence time in the light and dark chamber and the latent period of the first transition to the dark chamber were recorded for 3 minutes. Then, in a dark chamber, the rat received a single shock with a current of 1.5 mA through the electrode floor from a stimulator with a current output with digital indexation in milliamperes. Irritation was continued until the rat exited into the bright chamber through the transitional opening (training). A passive avoidance reaction test was performed 24 hours after training. It was found that heptapeptide at a dose of 300 μg / kg facilitated the production of passive avoidance reaction. Significantly (P <0.05) increased the latent period of animals entering the dark chamber and the total time spent in it during the entire testing period (3 minutes). When checking the safety of the reflex 24 hours after training, the number of animals that reached the learning criterion (> 300 s) in the experimental group was 151% of the control (P <0.05), which indicated better memorization of the experimental situation (Table 10).

 Example 7. The effect on the conditioned reflex of active avoidance (URAI).

 The development of a conditioned active avoidance reflex (URAI) was carried out using a shuttle camera manufactured by Ugo-Basile (Italy). The chamber consisted of two equal compartments, separated by a partition and a hole with a diameter of 9 cm. Unconditional electric skin (pain) irritation was supplied through the plates of the electrode floor to the legs of the animal. Each irritation lasts 4 seconds. The isolated action of the conditioned signals (light and sound) was 4 seconds, the interval between the presentation of the conditioned signals was 2 seconds. To avoid pain, the rat must learn to cross the adjacent compartment of the chamber within 4 seconds after turning on the conditioned signals. Current parameters: frequency 50 Hz, current strength 0.5-0.8 mA, depending on the individual severity of the response.

 URAI developed 25 combinations of stimuli daily for 4 days. The following were recorded: the number of conditioned avoidance reactions and the number of short-latent deliverance reactions (<1 s). The heptapeptide was administered intraperitoneally 15 minutes before each training session at a dose of 300 μg / kg.

 It was found that a heptapeptide at a dose of 300 μg / kg facilitated the development of URAI: on the third and fourth day of training, a significant (P <0.05) increase in the number of avoidances was observed compared with control animals. The number of short-latent deliverance reactions was also increased, which indicated the readiness of animals to perform this (targeted) activity (Table 11).

 Example 8. The effect on the conditioned food labyrinth reflex.

 The effect of the heptapeptide on the conditioned reflex with positive nutritional reinforcement was studied using a complex multi-path maze. Rats were previously trained to find a feeding trough with food. The training was carried out on animals with relative food deprivation for 4 days. Daily, the task of finding food was presented 5 times, the duration of each attempt did not exceed 5 minutes (otherwise the task was considered to be unfulfilled). As reinforcements used bread balls with a diameter of 10 mm. The following were recorded: the number of reactions performed on each day of the experiment and the latent period of their execution; number of errors, i.e. the number of entries into the dead ends of the labyrinth; the number of failed reactions (limitation - 5 minutes in the maze). Latent period of exit from the launch compartment. The heptapeptide was administered intraperitoneally repeatedly 30 minutes before each training session at a dose of 300 μg / kg.

 It was found that the heptapeptide has a significant and reliable facilitating effect on the formation of a complex conditioned food reflex, providing an acceleration of its formation. From the 2nd day of the training session in experimental animals there is a significant (P <0.05) increase in the number of completed food-producing reactions; the number of trained animals increases by 1.5–2 times (compared with the control group). The latent period of exit from the starting compartment of the labyrinth is progressively reduced with repeated injections of the heptapeptide.

 An extended experimental study of the action of the heptapeptide on learning and memory allows us to conclude that there is a pronounced positive effect on the mnestic processes associated with fixing consolidation and reproduction.

 In the above examples, the heptapeptide was administered predominantly as an aqueous solution. As mentioned above, the dosage form called Selank is a 0.15% aqueous solution of heptapeptide with the addition of 0.1% nipagin in 3 ml dropper bottles. The results obtained during the study of the Selank dosage form allowed us to determine the optimal dose range for the manifestation of the specific pharmacological action of Selank, which completely coincides with the above-described action of the heptapeptide.

 It was found that in the dose range of 150-400 μg / kg, a characteristic dose-response relationship is observed with an increase in activity to doses of 250-300-400 μg / kg and the establishment of plateau activity with a further increase in dose. It was established that Selank does not detect toxic effects when administered in doses 200 or more times higher than therapeutic.

 The following examples demonstrate the identity of the pharmacological action of the heptapeptide and Selank, which were obtained during the experiments performed on the same day, on the same batch of experimental animals in compliance with the standard experimental regimen.

 Example 9. Comparison of the action of heptapeptide and Selank on the anxiolytic effect in PKL tests in mice with passive and active ESR phenotype (Balb / c line and C57B1 / 6).

 As noted above, the selective anxiolytic effect is reliably detected in Balb / c mice, the behavior of which, when exposed emotionally in a stressful conflict situation (OP, PCL), is characterized by a freezing reaction.

 It was found that both an aqueous solution of heptapeptide and Selank dosage form have a pronounced selective anxiolytic effect in the PKL test. In mice with a pronounced freezing reaction and a high level of anxiety, the number of runs in the bright arms of the labyrinth, the total residence time in the bright parts of the labyrinth, and the ratio of the number of runs in the bright arms to the total number of movements along the arms of the labyrinth significantly increase. The latter indicator is the most important and generally accepted criterion characterizing the presence of anxiolytic properties of compounds (File, 1995). A ten-fold increase in the dose slightly increases the time spent by mice in the center of the maze and does not cause a decrease in motor activity or any other side effects (sedation, muscle relaxation, ataxia) (Table 12). The behavior of mice with the "active" ESR phenotype (line C57BL / 6) did not change significantly against the background of the studied compounds (Table 13).

 Example 10. Comparison of the effect of heptapeptide and Selank on anxiolytic action in tests of OP in Balb / c mice.

 The behavior of the Balb / c (c) line mice in the open field was initially characterized by the development of the freezing reaction and an extremely low level of locomotor activity, realized as horizontal movements along the periphery of the open field arena, without exits to the central zones. In this series of experiments, it was found that both the heptapeptide (Table 14) and Selank (Table 15) significantly increase overall motor activity, there are exits to the middle zones and even the intersection of the center of the open field arena, a freezing reaction does not occur. A high level of activity persists with increasing doses.

 Thus, in specific situations of OP and PCL used to detect the anxiolytic effect of known and new compounds, both the heptapeptide and the Selank dosage form activated the behavior of mice with a “passive type” ESR. The same doses of heptapeptide and Selank did not have a pronounced effect on the behavior of mice with an "active" type of ESD under the same conditions. The pharmacological effect of both studied forms (heptapeptide and Selank dosage form) can be presented, by analogy with benzodiazepine tranquilizers, as anxiolytic, tranquil activating, but not transient, but tranquil sedative.

 Example 11. Comparison of the action on the learning processes and memory of the heptapeptide and Selank.

 For a comparative assessment of the effect of the heptapeptide and the Selank dosage form on the learning and memory processes, we used the method of generating a conditioned reflex food-producing reaction in rats. The development of the reaction was carried out during one session during 30 trainings in a chamber 150x16x23 cm in size, having a start, central and target compartments. There was also food reinforcement in the target compartment. The movement of animals and the reaction time were recorded using photocells. Testing the preservation of skill was carried out after 24 hours, 7 days and 1 month. The substances were administered once at a dose of 300 μg / kg intranasally during a training session after the first 10 trainings (Table 16).

 It was found that both the introduction of the heptapeptide and Selank facilitates the formation of conditioned reflex motor food-producing reaction and its reproduction even 30 days after training. Both compounds cause a reduction in reaction time, most pronounced at the final stage of training: 40 seconds — control, 25 seconds — against the background of heptapeptide, 15 seconds — against the background of the Selank dosage form. The positive effect on the conditioned reflex food-producing reaction persisted 24 hours, 7 days and a month after training. Animals demonstrate a significantly higher level of skill retention, which is expressed by lower values of the time (speed) of the reaction in comparison with the control.

 Thus, both the heptapeptide and the Selank dosage form have an activating effect on the learning and memory of rats. At the same time, no differences in the nature of their activity were revealed.

 Experimental studies of Selank allow us to offer it as a selective anxiolytic that activates mnestic functions.

 Selank is non-toxic, convenient for use in clinical practice (nasal drops), with good drug tolerance.

 Comparison of Selank with modern tranquilizers revealed its significant advantages.

 The advantages of Selank are that it consists of natural amino acids found in the body and metabolizes rapidly. Based on the metabolism of peptides and amino acids, toxic products are not formed in the body. For Selank, it was not possible to detect any disturbances in the condition of the animals with an increase in the active dose of 200 or more times.

 Comparison of Selank with antipsychotics, tranquilizers, antidepressants showed the absence of Selank side effects characteristic of the latter (tab. 17-19).

Claims (2)

 1. The use of the heptapeptide Thr-Lys-Pro-Arg-Pro-Gly-Pro as an anxiolytic agent. 2. A pharmaceutical composition with anxiolytic action, containing the active substance and distilled water, characterized in that the active substance contains a heptapeptide of the formula Thr-Lys-Pro-Arg-Pro-Gly-Pro and an additional preservative substance nipagin in the following ratios of components g / l :
The heptapeptide of the specified formula is 1.45 - 1.55
Nipagin - 0.95 - 1.05
Distilled Water - Else

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