RU2529817C1 - 9-[2-(4-isopropylphenoxy)ethyl]adenine possessing antidepressant and stress-relieving action - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a therapeutic agent possessing antidepressant and stress-relieving action. The agent represents 9-[2-(4-isopropylphenoxy)ethyl]adenine distinguished for its anti-viral activity.

EFFECT: what is developed is psychotropic agent able to inhibit stress-inducing morphosomatic, cognitive and psychoemotional disorders, as well as possessing autonomous antidepressant activity.

14 tbl, 6 ex

Description

The invention relates to medicine, in particular to an adenine derivative having a pronounced antidepressant and anti-stress effect, which can be used to treat widespread neuropsychiatric diseases.

According to forecasts of the World Health Organization, by 2025–2030, mortality from depression will be on the first place in the world, ahead of cardiovascular, oncological and infectious diseases among other causes of death. Already, depression is one of the most serious public health problems. In the modern world, depression is becoming more common and more and more ongoing disorder. The high incidence of depression and their negative impact on the quality of life has been demonstrated in patients with various pathologies, including oncological diseases, HIV infection, viral hepatitis, skin diseases, rheumatoid arthritis, and peptic ulcer disease. In elderly patients, the combination of depression with somatic diseases is particularly problematic. Finally, depression is one of the main causes of suicide [Wittchen N., Jacobi F., Rehm J. The size and burden of mental disorders and other disorders of the brain in Europe 2010 // Eur. Neuropsychopharmacol. - 2011 .-- Vol.21. - No. 9. - P.655-679].

Despite the fact that a wide range of psychotropic drugs of various chemical groups has been created to date, the problem of pharmacotherapy for depression is far from being solved. Many of the known tranquilizers, antidepressants, and anxiolytics are characterized either by low therapeutic activity or by the presence of a significant number of undesirable side effects. For example, the appointment of the majority of modern antidepressants in the course of therapy in adequate therapeutic doses leads to a positive clinical effect in only 65-75% of patients. At the same time, antidepressants have a number of significant drawbacks: a long latent period of action, insufficient latitude and persistence of the therapeutic effect, a high likelihood of side effects, and the risk of toxic effects in case of an overdose. In this regard, the creation of psychotropic drugs with a multivalent effect, combining the antidepressant activity itself with the ability to positively affect the intellectual and mental functions and mental state of the patient, having a short latent period of action and sufficient therapeutic breadth, with a minimum of side effects, remains relevant.

In turn, stress, according to modern concepts, underlies a huge number of diseases and can lead to both new diseases and exacerbation of chronic diseases, cause somatic, neuropsychiatric disorders and multiple neuroendocrine disorders. The etiological factors in the pathogenesis of stressful lesions can be domestic, industrial, natural, technological, traffic accidents, terrorist acts or their threat, and much more [International Classification of Diseases, 10th revision (ICD-10). Classification of mental and behavioral disorders. - S.-P.: Overlayd, 1994]. According to the World Health Organization, annually more than 160 million people in the world are exposed to severe psycho-traumatic factors, resulting in various neuropsychiatric and psychosomatic pathologies [Ursano RS, Boldenberg M., Zhang I. Posttraumatic stress disorder and traumatic stress: from bench to bedside, from war to disaster // Ann. N.-Y. Acad. Sci. - 2010 .-- Vol. 1208. - P.72-81].

Antidepressants and anxiolytics are used to prevent and treat stressful diseases, however, in 50% of cases, such therapy does not provide the desired effect [Ravindran I.N., Stein M.V. Pharmacotherapy of post-traumatic stress disorder // Carr. Top Behav. Neurosci. - 2010 .-- Vol. 2. - P.505-525], which determines the need to continue the search for new, more effective pharmacological agents for the prevention and treatment of neuropsychiatric and psychosomatic pathologies associated with severe stressful effects.

The aim of the invention is the development of a new psychotropic drug with antidepressant action and capable of suppressing stress-induced morphosomatic, cognitive and psychoemotional disorders.

The invention consists in the use of 9- [2- (4-isopropylphenoxy) ethyl] adenine of the formula:

as an agent with antidepressant and anti-stress action.

9- [2- (4-Isopropylphenoxy) ethyl] adenine (laboratory code BMA-99-82) is a known purine derivative having high activity against human cytomegalovirus [Russian Patent No. 2233842 (2003) // Petrov V. I., Ozerov A.A., Novikov M.S., Pokrovsky V.I., Pokrovsky V.V., De Klerk E., Balzarini I. Derivatives of purine with antiviral activity (IPC C07D 473/34, A61K 31 / 52, A61P 31/12)]. When conducting in-depth preclinical studies of this compound in order to create an antiviral drug based on it, we revealed a pronounced psychopharmacological activity, in particular, an antidepressant and anti-stress effect.

The following methods were used to evaluate the anti-stress effect of 9- [2- (4-isopropylphenoxy) ethyl] adenine:

Assessment of somatic status of animals:

1. The study of the dynamics of body weight.

2. Monitoring the behavior of animals in the home cage with an assessment of the response to tactile, painful, sound stimuli.

Assessment of the emotional status of animals:

1. Test unavoidable forced swimming in Porsolt [Andreeva N.I. Guidelines for the study of the antidepressant activity of pharmacological substances // Guide to the experimental (preclinical) study of new pharmacological substances. - M .: IIA "Remedium", 2000. - S.121-126].

2. Consumption / preference test for sucrose solution (SHPS). [Sarkisova K.Yu., Folkomina A.A. The effect of a selective serotonin reuptake inhibitor fluoxetine on the symptoms of depressive behavior in rats of the WAG / RIJ strain // Zh. higher nervous activity - 2010 .-- T.60. - No. 1. - S.98-108].

3. The test "Open field" (OP) [Voronina TA, Seredenin SB Guidelines for the study of the tranquilizing (anxiolytic) action of pharmacological substances // Guide to the experimental (preclinical) study of new pharmacological substances. - M .: IIA "Remedium", 2005. - S.126-130].

4. Test “Elevated cross-shaped labyrinth” (PCL) [Voronina T.A., Seredenin S.B. Guidelines for the study of the tranquilizing (anxiolytic) action of pharmacological substances // Guide to the experimental (preclinical) study of new pharmacological substances. - M .: IIA "Remedium", 2005. - S.126-130].

5. Test for determining the threshold of provoked unmotivated aggression [Kozlovsky V.L., Prakhie I.V. The effect of calcium channel blockers on the effects of antidepressants // Experiment. the clinic. pharmacol. - 1996. - T.59. - No. 5. - S.9-11].

Assessment of cognitive functions:

1. Test "conditional reaction of passive avoidance" (passive avoidance reaction) [Voronina T.A., Ostrovskaya R.U. Guidelines for the study of the nootropic activity of pharmacological substances // Guide to the experimental (preclinical) study of new pharmacological substances. - M.: Publishing House "Medicine", 2005. - S.295-308].

2. Test "extrapolation deliverance" (TEI) [Voronina TA, Ostrovskaya R.U. Guidelines for the study of the nootropic activity of pharmacological substances // Guide to the experimental (preclinical) study of new pharmacological substances. - M .: OJSC "Publishing house" Medicine ", 2005. - S.295-308].

Assessment of the severity of stress-related morphosomatic changes:

1. Determination of the degree of involution of lymphoid organs - thymus and spleen.

2. Determining the degree of adrenal hypertrophy.

3. Determining the degree of ulceration of the gastric mucosa.

The following examples illustrate the invention.

Example 1. The effect of 9- [2- (4-isopropylphenoxy) ethyl] adenine on the somatic status of stressed animals.

To assess the antistress effect, a model of the combined effect of several stressful factors was chosen. Such a model makes it possible to simultaneously obtain impaired somatic status in animals, a pronounced Selye triad — involution of lymphoid organs (thymus and spleen), adrenal hypertrophy and ulceration of the gastric mucosa, as well as disturbances in the psychoemotional state (anxiety-depressive, aggressive) and cognitive function. The study used a rigid model with immobilization and alternating exposure to loud sound, pulsating light and vibration, which, of course, is an impact of an extremely threatening or even catastrophic nature that can cause distress in any experimental animal. The antidepressants imipramine and sertraline and anxiolytic with the nootropic effect of phenibut were used as reference drugs.

Modeling of stress injuries in experimental animals was carried out in a setup representing a dark chamber 28 cm wide, 36 cm long and 28 cm high, divided into 6 isolated compartments of the same volume and designed for simultaneous stressing from 1 to 6 experimental animals, mounted on an inclined angle of 10 ° flat metal platform. The platform is equipped with an electric drive making oscillating movements with an adjustable frequency from 1 to 50 vibrations per minute. The chamber is equipped with an opaque cover; ventilation holes are provided around the perimeter of the chamber for air circulation. The lid of the camera is equipped with a sound noise source and 3 light sources located at a height of 28 cm. The light sources are equipped with a relay interrupter with a response time of every 2 s, which makes it possible to obtain pulsating light. Rats are placed in isolated compartments and exposed to various stressful factors for 30 minutes: pulsating light, loud sound and vibration. The installation allows the combination of several stressful stimuli. To simulate severe stress disorders in animals of somatic, neuropsychiatric and cognitive status in animals, they underwent prolonged chronic unavoidable stress for 10 days (daily for 30 minutes) with the change of multimodal stress stimuli every 5 minutes. The stimuli are changed according to the stachostic scheme so that each subsequent stressful effect is unpredictable for animals. For example, on the first day of stressing, the sequence of stimuli is as follows: the first 5 min - vibration, the second 5 min - a sharp loud sound, the third 5 min - pulsating light, the fourth 5 min - vibration and sound, the fifth 5 min - vibration and light, sixth 5 min - sound and light. Then, on the second day of stressing, irritants are applied, starting from the one that was second on the first day, on the third day - from that which was the third on the first day and so on. Under such conditions of prolonged unavoidable unpredictable stressful effects for animals, conditions of emotional stress and frustration are created, leading to the formation of despair, terminologically defined as “learned helplessness” and manifested in the development of persistent anxiety-depressive state, impaired cognitive function, pronounced morphosomatic changes. A total of 6 groups of 8 animals were used - 48 animals (male rats). The test compound and comparison drugs were administered to animals against the background of already developed neuropsychiatric, cognitive and morphosomatic lesions with the first injection 24 hours after the last stress with a 10-day course (once daily, orally) in the following doses: VMA-99-82 - 30 mg / kg, imipramine - 15 mg / kg, sertraline - 10 mg / kg and phenibut - 25 mg / kg. The indicated doses are most effective according to the results of previous experimental studies and based on an analysis of the literature. Assessment of behavioral disorders in animals using psychopharmacological tests was performed after a single injection of substances, and then again after a 10-day course of administration of substances (tests were performed 45 minutes after the last injection).

During each series of experiments, the dynamics of changes in body weight in animals of the experimental and control groups were recorded by daily weighing on an electronic balance at the same time, immediately before the administration of the studied compound, during the entire observation period. Then calculated the average body weight of the animals in groups. In addition, visually recorded indicators of the general somatic status of animals were taken into account daily:

1. General condition, appearance (condition of the mucous membranes - color, swelling, presence and nature of secretions; condition of the coat - the hair is ruffled or smooth, shiny or dull); the behavior of animals in the home cage (activity, the presence of aggressive attacks, the presence of auto- and allogrooming).

2. Neuromuscular irritability: reaction to external stimuli - sound (tapping on the cell), tactile (touch), pain (compression of the tip of the tail).

3. The functional state of the autonomic nervous system: ptoses, especially urination and defecation, salivation.

4. Threshold of pain sensitivity in animals by paw irritation by electric current.

The results summarizing the general somatic state of stressed animals are presented in Tables 1 and 2.

Table 1 Dynamics of body weight in stressed animals Groups of animals Body weight (g) 1 day of administration 2 day introduction 3rd day of administration 4th day of administration 5th day of administration 6 day introduction 7 day introduction 8 day introduction 9th day of administration Intact control 329 ± 18 321 ± 16 319 ± 15 316 ± 16 317 ± 14 318 ± 11 320 ± 14 324 ± 14 329 ± 14 Control + stress 285 ± 30 270 ± 23 274 ± 21 276 ± 20 282 ± 24 288 ± 25 288 ± 23 289 ± 24 292 ± 26 VMA-99-82 288 ± 11 308 ± 12 315 ± 5 317 ± 16 318 ± 13 324 ± 15 327 ± 15 332 ± 15 327 ± 14 Imipramine 278 ± 17 274 ± 17 268 ± 16 264 ± 13 271 ± 112 274 ± 14 283 ± 14 283 ± 14 286 ± 13 Sertrlin 282 ± 17 268 ± 15 264 ± 19 267 ± 19 275 ± 18 285 ± 16 289 ± 15 297 ± 16 301 ± 16 Phenibut 302 ± 23 305 ± 26 304 ± 23 309 ± 18 311 ± 24 316 ± 23 314 ± 29 31 ± 27 320 ± 22

table 2 The threshold of pain sensitivity in stressed animals on the background of the introduction of the connection VMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Imipramine Sertraline Phenibut Vocalization threshold, B 30.3 ± 2.4 22.7 ± 2.2 # 32.7 ± 2.8 * 28.3 ± 2.0 25.3 ± 2.1 29.6 ± 2.0 Note: hereinafter, # - p <0.05; ## - p <0.01 - the statistical significance of the differences compared with the indicator of the group of animals "Control intact" of the corresponding day of the study; * - p <0.05; ** - p <0.01 - statistical significance of the differences compared with the parameter of the Control + Stress group of animals of the corresponding study day (Kruskal-Wallis rank one-way analysis, Newman-Keyles test).

Chronic 10-day stress exposure led to a deterioration in the general condition of animals of all experimental groups. Upon visual examination after stressing, the skin and mucous membranes of rats were physiological in color, the reflexes were alive, however, the coat looked untidy, was tousled. The behavior of rats was characterized by general nonspecific arousal and neurotization. In animals exposed to chronic stressful effects, frequent auto- and allobarbering (characterizing stress in rodents) was observed, as a result of which patches of focal allopecia were observed on the coat of rats (most often on the muzzle around the nose, sides and dorsal surface of the neck, some individuals were gnawed part of vibrissae). Animals reacted to the capture and movement of domestic cells by vocalization, locomotor agitation, the adoption of defensive and aggressive postures, and aggressive attacks on cell gratings. Reactions to external stimuli reflected the phenomena of arousal and hypersensitivity: rats reacted to tactile, pain and sound irritation with a sharp start, vocalization and attempts to attack the experimenter. Aggressiveness of stressed rats was manifested even without the presentation of external stimuli - when observing the behavior of rats in domestic cells in animals, confrontations were often observed. In addition, the anxiety and excitement of animals was expressed in the commission of frequent uprights and short-term grooming. The condition of the animals at the time of the end of the chronic stress exposure can be assessed as "moderate."

Intact control rats throughout the experimental series with the simulation of “learned helplessness” maintained a satisfactory condition, normal behavioral activity, and an adequate response to external stimuli. The appearance of the animals of this group was neat, they had a preserved motivation for the behavior of washing and cleaning (grooming). The color of the skin and mucous membranes, corneal reflexes, photoreaction of the pupils to light, the frequency and nature of urination and defecation in these animals did not have obvious pathological changes. The woolen coat looked neat, often there was a long “comfortable” auto-grooming.

The course therapeutic introduction of the studied substance contributed to the leveling of the phenomena of neurotic animals. In animals treated with the BMA-99-82 compound, the severity of excitation and hyperreactivity against external stimuli gradually decreased in the dynamics of the study. Animals less often vocalized, less aggressive attacks and poses were observed. Behavioral activity in the cell normalized, locomotor activity decreased, prolonged grooming appeared. By the 10th day of administration of the test substance, the animals began to look more neat, their condition returned to normal and was satisfactory.

Animals that were injected with the connection BMA-99-82, were less pronounced excitement and aggressiveness compared with the control stress group of animals. At the end of stress, they did not show aggressive attacks in the cell and looked more neat when applied to external stimuli. Their response to tactile, sound, and pain stimuli was virtually identical to that of intact control rats. Less severe stress-related symptoms in animals of this group may be a consequence of the anti-stress action of the studied substance.

In this series of experiments in animals that received a course therapeutic introduction of reference substances with antidepressant activity - imipramine, sertraline and phenibut, various variants of body weight dynamics were noted (Table: 1). The animals of all these experimental groups during the process of repeated acts of stress lost an average weight of 13-15% compared with the initial indicators, which is a consequence of chronic stress. In animals of the Control + Stress group, from the second day after the cessation of stress, the mass gradually increased, which reflects an improvement in the condition and a decrease in the manifestations of stress, however, until the last day of observation, it remained statistically significantly lower than in animals of the intact control. In rats treated with imipramine and sertraline, in the first three to four days after the end of the stress exposure, body weight decreased moderately, and by the fourth day it stabilized and began to grow from 5-6 days, and after the cessation of stress, the average weight indicators in animals of these groups were already did not differ from indicators of intact control. In animals that were treated with phenibut, as in the case of BMA-99-82, stable increases in body weight in dynamics were observed over the entire observation period.

In the group of animals “Control + stress”, a decrease in the threshold of pain sensitivity was statistically significant compared with intact rats, which may be a consequence of the general neuroticism of animals and an increase in the emotional response to pain (Table 2). In animals treated with BMA-99-82 and imipramine once, the threshold of pain sensitivity was statistically significantly higher than that of the Control + Stress group, which may be the result of the anti-stress effect of the substances.

Example 2. The effect of 9- [2- (4-isopropylphenoxy) ethyl] adenine on the emotional state of stressed animals.

Test of forced unavoidable swimming in Porsolt. The unit for performing the test is a cylinder made of transparent acrylic plastic with a diameter of 20 cm and a height of 45 cm (manufacturer Open Open Science LLC, Moscow, Russia). The classic version of the test is carried out in 2 stages. At the 1st stage, intact animals undergo stress without the introduction of the studied compounds. Rats were placed for 15 min in a container filled with water with a temperature of 17-19 ° C to such a level that the animals could not rest with their hind legs and tail against the bottom. The 2nd stage begins after 24 hours with the introduction of the test substances taking into account the peak of their action, then the animals are again placed in a container with water under the same conditions as on the first day, recording indicators of depressive behavior for 300 s: latent period of immobilization ( the rate of development of a depressive state); the time spent by rats in a state of immobilization (animals passively swim in water with their heads slightly raised) is a behavioral correlate of despair, one of the pathognomonic manifestations of anxiety and depression caused by exhaustion due to stress exposure. An increase in the latent period of immobilization, a decrease in its duration, a decrease in the duration of passive swimming, an increase in the duration of active swimming and the number of jumps under the influence of the test compound compared with the control group is regarded as having pronounced anti-stress properties.

Test of consumption / preference of sucrose solution (P / PS). The sucrose / water alternative choice test has been adopted to evaluate the enjoyment behavior of animals. Animal consumption of a sweet 20% sucrose solution is a hedonic indicator widely used to assess the sensitivity of animals to reinforcements that cause a feeling of pleasure. To perform the test, animals are placed for 1 h one at a time in cages (after preliminary 8-hour water and food deprivation), in which, under free choice conditions, they are presented with two drinkers: one with a 20% sucrose solution, the other with ordinary water. The consumption of sucrose and water is defined as the difference in the weight of the drinkers before and after testing. The coefficient of sucrose consumption (CPS) in g / 100 g of the animal’s body weight is also calculated by the formula: CPS = actual sucrose consumption (g) × 100 / actual animal weight (g). In addition, assess the preference for sucrose (PS) in% of the total fluid intake by the formula: PS = sucrose intake (g) × 100% / total fluid intake (sucrose + water) (g). Reduced sucrose intake is indicative of signs of anhedonia, which is a reliable indicator of depressed animals due to stress or other damaging factors. A decrease in total fluid intake is seen as a result of the suppression of one of the vital motivations. An increase in the consumption and preferences of sucrose, as well as the preservation of the motivation for quenching thirst when modeling stressful effects on animals, is an indicator of the anti-stress effect.

Open Field Test (OP). The open-field technique is used to study the effect of a substance on indices of spontaneous individual behavior of animals (motor, orientational-research activity and emotional status), and allows to reveal violations of their psychoemotional state. The experiment is carried out in a round arena with a diameter of 97 cm and a wall height of 42 cm. The floor of the chamber is divided by black paint into 25 equal squares measuring 5 × 5 cm with the allocation of the central zone of the field. The chamber floor is equipped with 16 holes with a diameter of 2 cm (manufacturer Open Open Science LLC, Moscow, Russia). The chamber is illuminated by a lamp with a power of 90 Lx located above the center of the field at a height of 150 cm. The rat is placed in the center of the chamber with its tail to the experimenter and its behavior is observed for 3 minutes. In the test, the following are recorded: the latent period of exit from the center of the field, the number of crossed squares on the periphery, the number of crossed brightly lit squares, the number of vertical stands - parietal and free, the number of peeps in holes, the number of urinations and bowel movements, the number of acts of short-term (up to 5 s) grooming . The latent period of exit from the center of the field (the time from the moment the animal was placed in the test setup unit to the exit from the central zone of the field) is an indicator of the rate of approximate reactions, the number of crossed squares characterizes the spontaneous locomotor activity of animals, the sum of racks and peeps in the holes is the total estimated research activity. Parietal racks are an indicator of anxiety behavior, alertness, free racks characterize calm research behavior, the less animals make parietal racks and the more free, the lower their anxiety level. The number of visits to the central brightly lit area (aversive for burrowing rodents) also indicates the level of anxiety of the animal (the less anxiety - the more, and vice versa). The behavior of the animal when washing and brushing (grooming) reflects the degree of its emotional stress: frequent short-term (up to 5 s) anxiety state, rare and long-lasting state of comfort. Vegetative indicators of stress (the number of boluses for bowel movements and urination) also characterize the emotional stress of animals. A decrease in the number of parietal racks, acts of short-term grooming, the number of boluses of defecation and urination in combination with an increase in the number of free racks, visits to the center of the field in animals receiving test substances, compared with the group of control stressed animals, indicates its anti-stress effect.

Test "Elevated Cruciform Maze" (PCL). The technique of an elevated cruciform labyrinth is used to study the effect of a substance on the severity of the emotional reaction of fear and anxiety in test animals and to identify antistress and / or anxiolytic activity. It is based on the preference by rodents of dark holes, the natural fear of being in open areas and falling from a height. Under stress exposure, depending on its strength and duration, an increase in anxiety and fear can be observed in animals. The experiment is carried out in an installation (labyrinth) of non-laminated gray polyvinyl chloride, consisting of a central platform 14 × 14 cm in size, from which four arms 50 × 14 cm each diagonal crosswise at right angles. Two opposite sleeves - open with a side height of 1 cm, and two - closed, dark, bounded by sides with a height of 30 cm. The labyrinth is mounted on the cross of an aluminum trolley 55 cm high, 100 cm wide (manufacturer Open Open Science, Moscow, Russia ) The test animal is placed on the central area of the maze with its head to the open sleeve and is monitored for 3 minutes. In the test are recorded: the time spent by the animals on the central platform, in open and closed sleeves, the number of racks in closed and open sleeves, the number of hangings from open sleeves and the number of entries into the central zone, open and closed sleeves. The rat crossing the borders of any compartment with its hind legs was defined as the entry of the animal into the labyrinth compartment. An increase in the residence time in light arms, the number of entries in them, the uprights in open arms and hanging from them indicates the presence of an anti-stress and anxiolytic effect in the test substance. The length of stay at the central site allows you to evaluate the speed of decision making animals. Total motor activity is estimated by the total number of entries into open, closed arms and the center of the maze.

Test for determining the threshold of provoked unmotivated aggression. The severity of the aggressive behavior of rats was assessed in a pair of animals formed randomly. Rats were planted in pairs on the electrode floor of a standard chamber made of organic glass measuring 27.5 × 27.5 × 40 cm, to which a voltage of gradually increasing amplitude was applied with a stimulator, starting at 20 V. The voltage was applied in periods of 3 s, separated by pauses of 1 from. If, after three presentations of a stimulus of the same intensity, aggressive reactions do not occur, the voltage increases by 1 V and the stimulation continues until aggression occurs in response to at least three impulses of the same force. This voltage is considered threshold. The registration of the voltage that provoked a fight in a pair of animals is an objective criterion that reflects the threshold of provoked aggression. An aggressive reaction is considered to be the behavior of rats in which they both stand on their hind legs with their faces toward each other and try to strike with their front and hind limbs and bite each other. For substances with anxiolytic and antidepressant action, an increase in the threshold of aggressiveness is characteristic - anti-aggressive effect.

Summary data on the effect of the BMA-99-82 compound and comparison drugs on the emotional status of stressed animals (with “learned helplessness”) are presented in Tables 3-10.

The Porsolt test in animals of the Control + Stress group showed a pronounced depressive state: the latent period of immobilization and indicators of active avoidance of an aversive situation decreased statistically significantly - the number of jumps and the duration of active swimming, as well as increased immobility time (Tables 3 and 4). Compound ВМА-99-82, as well as all the studied comparison preparations, statistically significantly reduced the manifestations of a depressed state in animals - increased the latent period of immobilization, the number of jumps and the time of active swimming, and reduced the duration of immobility. According to the severity of this effect, the compound BMA-99-82 was comparable with sertraline and slightly superior to imipramine and phenibut. It should be noted that in this model of depression, the effectiveness of all studied substances increased against the background of their course therapeutic use. The effect of the BMA-99-82 compound on most indicators of depressive behavior during repeated testing after course use ceased to be statistically significant, which may be a consequence of a decrease in the severity of the depressive state in the control group of animals (“Control + stress”).

Table 3 The severity of depressive behavior in the Porsolt test in stressed animals on the background of the introduction of VMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Imipramine Sertraline Phenibut 1x introduction Latent period of immobilization, s 96.7 ± 6.6 53.2 ± 4.5 75.8 ± 6.2 * 77.1 ± 6.5 * 63.5 ± 5.6 66.2 ± 8.1 Duration of immobilization, s 44.0 ± 4.0 101.3 ± 7.8 77.5 ± 5.5 * 59.3 ± 4.8 ** 74.3 ± 5.8 * 83.0 ± 7.6 ** 10 day introduction Latent period of immobilization, s 89.5 ± 8.1 74.8 ± 6.6 91.2 ± 6.3 125.3 ± 9.4 ** 96.2 ± 6.3 * 90.3 ± 8.8 * Duration of immobilization, s 49.5 ± 4.1 81.5 ± 5.2 ## 64.1 ± 5.0 * 47.3 ± 3.5 ** 61.4 ± 5.0 73.2 ± 5.8 **

The results of the study showed that animals with "learned helplessness" develop a pronounced suppression of hedonic motivation, characteristic of depression, expressed in a statistically significant decrease in the coefficient of consumption and percent preference for a 20% sucrose solution (Table 5). Compound BMA-99-82, as well as all comparison drugs, statistically significantly increased the consumption and preference for sucrose in animals, which also indicates the presence of an antistress effect. According to the severity of this effect, the compound BMA-99-82 was comparable with sertraline, imipramine and phenibut. The pronounced antidepressant effect of the studied substance on the model of stress-induced depression “learned helplessness” can be a consequence of both the antidepressant effect itself and its anti-stress activity.

Table 4 The severity of active behavior of avoiding an aversive situation in the Porsolt test in stressed animals against the background of the introduction of BMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Imipramine Sertraline Phenibut 1x introduction Number of jumps 10.8 ± 0.9 4.7 ± 0.8 ## 8.0 ± 0.7 * 9.5 ± 0.6 ** 6.3 ± 0.8 7.9 ± 0.6 Duration of active swimming, s 138.7 ± 9.4 79.3 ± 5.4 # 120.2 ± 9.3 ** 153.5 ± 10.2 ** 108.3 ± 8.7 * 132.0 ± 11.0 * 10 day introduction Number of jumps 8.7 ± 0.9 7.2 ± 0.8 6.8 ± 0.7 11.5 ± 0.8 ** 10.2 ± 0.8 * 6.5 ± 1.0 Duration of active swimming, s 122.5 ± 8.6 95.3 ± 7.3 # 115.7 ± 10.7 166.5 ± 10.8 ** 153.5 ± 11.4 ** 119.0 ± 9.6

In the Open Field test, chronic stress and the formation of “learned helplessness” led to an increase in the level of anxiety in animals of the control group (“Control + stress”), which was expressed in a statistically significant decrease in the number of visits to the center, an increase in the number of wall racks, and acts of short-term grooming and fecal boluses in animals (Table 6). All the studied comparison drugs after a single and to a greater extent after a course of administration increased the number of visits to the center in animals, reduced the number of parietal stands, acts of short-term ("disturbing") grooming and fecal boluses in animals - suppressed the manifestations of anxiety caused by stress exposure. In this test, anxiolytic properties were most pronounced in phenibut.

Compound ВМА-99-82 also reduced the severity of anxiety manifestations in animals with "learned helplessness" - showed moderate anxiolytic properties, while it had a statistically significant effect on anxiety indices in the "Open Field" during the first test after a single injection. When re-testing in the Open Field after a course of administration, the test indicators in animals treated with BMA-99-82 did not have statistically significant differences with both the intact control and the Control + Stress group.

Table 5 The severity of hedonic motivation in the test of consumption / preference of sucrose in stressed animals against the background of the introduction of BMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Imipramine Sertraline Phenibut 5 day introduction Sucrose consumption coefficient (g / 100 g body weight) 4.7 ± 0.6 1.3 ± 0.2 ## 3.7 ± 0.5 ** 2.8 ± 0.6 * 4.2 ± 0.3 ** 3.4 ± 0.3 Consumption (preference) of sucrose in% of total fluid intake 82.3 ± 4.1 36.2 ± 3.5 ## 55.5 ± 3.4 ** 48.7 ± 3.2 * 54.7 ± 2.8 ** 49.0 ± 3.1 9 day introduction Sucrose consumption coefficient (g / 100 g body weight) 3.5 ± 0.4 2.2 ± 0.4 # 3.3 ± 0.5 3.8 ± 0.6 * 3.5 ± 0.3 * 3.5 ± 0.4 Consumption (preference) of sucrose in% of total fluid intake 68.0 ± 4.3 47.7 ± 3.9 ## 63.5 ± 4.1 ** 65.5 ± 5.1 * 77.2 ± 4.3 ** 58.0 ± 5.2

Table 6 The severity of locomotor and research behavior in the "Open field" in stressed animals after the introduction of VMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Sertraline Imipramine Phenibut The number of crossed central squares 30.5 ± 2.2 15.5 ± 1.3 ## 24.0 ± 1.8 ** 19.2 ± 1.6 20.2 ± 1.2 * 20.2 ± 1.2 The number of intersected parietal squares 6.5 ± 0.7 12.2 ± 0.7 ## 6.8 ± 0.5 6.7 ± 0.8 8.7 ± 0.4 * 7.3 ± 0.6 Number of visits to the center 1.5 ± 0.2 0.3 ± 0.2 ## 0.7 ± 0.2 0.8 ± 0.2 0.8 ± 0.3 1.2 ± 0.3 The number of wall racks 3.7 ± 0.3 5.2 ± 0.5 # 3.5 ± 0.3 * 3.7 ± 0.3 * 4,5 ± 0,4 3.3 ± 0.3 The number of acts of short grooming 2.7 ± 0.3 7.2 ± 0.6 ## 5.0 ± 0.5 * 5.3 ± 0.5 * 5.8 ± 0.7 3.9 ± 0.9 Fecal bolus count 2.7 ± 0.3 5.8 ± 0.5 ## 2.5 ± 0.3 ** 4,5 ± 0,4 5.0 ± 0.6 2.7 ± 0.7

In animals treated with compound ВМА-99-82, in comparison with the control group, there were more exits to the central zone, a greater number of crossed central squares and significantly fewer parietal stands, crossed peripheral squares (Table 6), the number of acts of short-term grooming and fecal boluses . The data obtained indicate that the connection BMA-99-82 reduces the phenomenon of distress, which is clearly observed in animals of the control group subjected to chronic combined stress.

In the “Elevated Cruciform Maze” in stressed animals of the control group (“Control + stress”), pronounced behavioral disturbances were also observed that characterized high anxiety: the time spent in open arms was statistically significantly reduced, the number of visits and hanging from open arms increased, the number of peeps increased from closed sleeves (risk assessments) (Table 7). Compound ВМА-99-82 (mainly when testing after a single use), as well as all comparison drugs (to a greater extent when testing after a course of administration) reduced the severity of the listed manifestations of anxiety in animals. According to the degree of prevention and elimination of alarming symptoms in this test, the BMA-99-82 compound was slightly inferior to phenibut and was comparable to imipramine and sertraline.

In addition to the above, when assessing the emotional status of stressed animals of the Control + Stress group in the test of unmotivated aggression provoked by electric pain stimulation, a statistically significant decrease in the threshold of an aggressive reaction was observed, which is consistent with the results of visual observations of the behavior of animals in the home cage (Table 8) . Compound ВМА-99-82, as well as all comparison preparations increased this indicator, i.e., they suppressed aggressiveness in animals. In terms of the severity of this effect, the studied substance was superior to sertraline and phenibut and was comparable to imipramine.

Table 7 The severity of behavioral manifestations of anxiety in the "Elevated Cruciform Maze" in stressed animals on the background of the introduction of VMA 99-82 and comparison drugs Registered
indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Sertraline Imipramine Feniote 1x introduction Duration of stay in open sleeves, s 37.7 ± 4.0 14.5 ± 1.6 ## 30.2 ± 1.8 * 17.7 ± 1.3 19.5 ± 1.3 * 23.3 ± 1.6 Number of entries in open sleeves 2.7 ± 0.3 1.3 ± 0.2 ## 2.2 ± 0.2 * 2.2 ± 0.3 * 1.7 ± 0.3 1.6 ± 0.2 Number of hangings from open sleeves 2.5 ± 0.4 0.3 ± 0.2 ## 1.8 ± 0.5 * 0.8 ± 0.4 1.7 ± 0.5 * 2.1 ± 0.3 Number of peeks from closed sleeves 2.7 ± 0.3 5.5 ± 0.4 ## 4.8 ± 0.5 4,5 ± 0,6 5.0 ± 0.4 3.9 ± 0.4 10 day introduction Duration of stay in open sleeves, s 49.2 ± 3.8 32.0 ± 3.0 ## 51.5 ± 3.5 57.7 ± 5.4 ** 46.3 ± 4.4 * 43.2 ± 3.3 Number of entries in open sleeves 3.3 ± 0.3 1.8 ± 0.2 ## 2.3 ± 0.3 2.8 ± 0.3 * 2.3 ± 0.4 2.1 ± 0.2 Number of hangings from open sleeves 3.8 ± 0.5 2.3 ± 0.4 # 3.2 ± 0.3 2.8 ± 0.5 4.9 ± 0.5 ** 4.1 ± 0.5 Number of peeks from closed sleeves 2.2 ± 0.3 3.7 ± 0.3 ## 3.1 ± 0.4 2.8 ± 0.4 3.0 ± 0.5 2.9 ± 0.3

Table 8 The threshold of provoked unmotivated aggression in stressed animals on the background of the introduction of BMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Sertraline Imipramine Phenibut 4 day introduction Threshold of an aggressive reaction, V 38.1 ± 2.9 20.4 ± 1.5 ## 31.3 ± 2.6 ** 25.3 ± 1.1 * 26.7 ± 1.1 * 28.3 ± 1.8 * 9 day introduction Threshold of an aggressive reaction, V 31.0 ± 1.6 26.8 ± ± 1.7 34.3 ± 2.1 * 35.7 ± 1.6 * 35.3 ± 2.0 * 32.6 ± 3.1

Example 3. The effect of 9- [2- (4-isopropylphenoxy) ethyl] adenine on the cognitive function of stressed animals.

Two methodological approaches were used to study cognitive functions: the test of the conditioned reaction of passive avoidance and the test of extrapolation deliverance.

Test "Conditional reaction of passive avoidance" (passive avoidance reaction). The animals were trained in an installation consisting of two adjacent compartments, one of which is 60 × 40 × 40 cm in size, open at the top and brightly lit (90 Lux). Another camera with an electrified floor of a smaller size (15 × 15 × 15 cm) is darkened, closed on all sides and has an opening (8 × 8 cm) for communication with a large compartment. The experiment was carried out in two stages: at the first stage, the animals develop a conditioned reflex of passive avoidance, at the second stage, after 24 hours, the preservation of the memorial trail is determined. During training, the test animal is placed in the middle of the illuminated compartment area with its tail toward the hole in the dark compartment. Within 3 minutes, the animal is under visual observation. After each entry into the dark compartment, the animal is subjected to electric pain stimulation (40 B, 3 pulses of 1 s, with an interval of 0.5 s). Animals that did not enter the dark chamber during this time were excluded from the experiment. At the stage of animal training, the following indicators are recorded: the time from the moment the animal was placed in the middle of the site until the first entry into the dark compartment (the latent period of the first entry) and the number of entries into it. Determination of the safety of the memorial trail is carried out in the same way as in the first stage of the development of the conditioned reflex of passive avoidance: visual observation of the trained animal placed in the same installation is carried out for 3 minutes, however, if the animal enters the dark compartment, electric pain stimulation is not performed. The experiment compares the latent period of the first entry of animals into the dark compartment and the number of entries into it when playing a commemorative trace with similar indicators during training. An increase in the latency period of the first entry into the dark compartment, a decrease in the number of entries into it, as well as a decrease in the total time spent in the dark compartment, recorded during the play of the skill, indicate that the animal is trained and that information about the aversivity of the dark compartment is stored in memory.

Test "Extrapolation deliverance" (TEI). The animals were trained in a cylindrical plastic container (diameter 35 cm, height 45 cm) equipped with a removable inner cylinder made of transparent acrylic plastic (diameter 9 cm, height 23 cm), fixed in the center of the external container using special fasteners (manufacturer of Open Science LLC ", Moscow, Russia). The external container is filled with water with a temperature of 16-18 ° C to such a level that the edges of the inner cylinder are 2.5 cm immersed in it. The experiment is performed in two stages: at the first stage, the animals develop a conditioned reflex of getting rid of the aversive medium , at the second stage, after 24 hours, the safety of the skill of extrapolation disposal is checked. During training, the test animal was placed in an aquarium under a cap. For 3 minutes he was visually observed. Animals dive beneath the edges of the cap and thus get rid of the aversive aquatic environment. Animals that do not solve the problem are excluded from the experiment. At the stage of animal training, the following indicators are recorded: the time from the moment the animal is immersed in water until the active swimming begins (latent period of motor activity), immobilization time (the animal passively swims in the water with its head slightly raised, all four limbs are motionless), the number of jumps in the cap ( active attempts to jump out of the water) and the time from the moment the animal is immersed in water until diving under the edges of the cap (latent period of diving). At the playback stage, indicators are recorded for 3 minutes in the same way. The experiment compares the indicators that determine the degree of training of the animal and the state of memory: the latent period of diving of the animal during training and reproduction of the extrapolation ridding skill (the more it decreases during the reproduction of the skill, the more trained the animal and the memorable footprint is preserved). In animals subjected to stress, especially severe and repeated many times, there is not only an anxiety-depressive state, but also a decrease in cognitive function.

The results of the assessment of cognitive functions in stressed animals are presented in tables 9 and 10.

Animals exposed to prolonged stressful effects (the Control + Stress group) showed marked cognitive impairment in the passive avoidance reaction and TEI tests: a statistically significant decrease in the latent period of the first entry into the dark compartment in the passive avoidance reaction and an increase in the latent diving period in the TEI during reflexes. The combination of BMA-99-82 and sertraline contributed to the improvement of learning and memory in animals in passive avoidance reaction, however, no statistically significant improvement in cognitive functions was noted with their use.

Table 9 Learning and memory in stressed animals in the test “Conditional passive avoidance reaction” against the background of the introduction of VMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Imipramine Sertraline Phenibut Reflex stage Latency of the first approach 23.5 ± 3.7 83.3 ± 3.3 # 49.0 ± 5.3 * 84.5 ± 1 * 67.8 ± 5.6 * 54.1 ± 7.1 * Number of calls 0.7 ± 0.2 0.8 ± 0.2 0.8 ± 0.3 0.2 ± 0.2 0.7 ± 0.2 0.7 ± 0.3 Playback after 24 hours (after a single injection) Latency of the first approach 158.8 ± 13.9 100.2 ± 25.3 143.5 ± 23.2 126.8 ± 23.9 127.7 ± 20.5 130.0 ± 14.2 Number of calls 0.8 ± 0.3 0.3 ± 0.2 0.3 ± 0.2 0.8 ± 0.4 0.5 ± 0.3 0.3 ± 0.3 * The number of animals entering the dark compartment,% 17 67 # 33 * fifty* 33 * 33 * Playback (10-day introduction) Latency of the first approach 132.9 ± 21.1 71.8 ± 21.7 139.2 ± 27.3 103.7 ± 24.3 129.2 ± 23.0 130.2 ± 16.3 * Number of calls 0.7 ± 0.3 1.7 ± 0.4 0.6 ± 0.4 1.3 ± 0.5 1.0 ± 0.5 0.6 ± 0.4 * The number of animals entering the dark compartment,% fifty 83 # fifty* 67 * fifty* fifty*

The tendency to a decrease in the severity of cognitive impairment in animals on the background of the administration of these drugs may be associated with their positive effect on the emotional status, which also matters in the formation of a memory trace. In stressed animals with “learned helplessness” receiving the test substance, more active behavior in the extrapolation interaction test was observed compared with animals in the control group (“Control + stress”). They quickly found deliverance from the aversive environment (diving faster) both during the training period and during reproduction after a single and ten-day introduction. According to the indicator “Effect of substances on the time of extrapolation,” the BMA-99-82 compound exceeded all comparison drugs: imipramine, sertraline and phenibut. At the same time, imipramine had a negative effect on the production and reproducibility of conditioned reflexes of avoiding the aversive factor.

Table 10 The effect of VMA-99-82 and comparison drugs on the time of “extrapolation disposal” Recorded Indicators (M ± m) Experimental groups Intact control Control + stress VMA-99-82 Imipramine Sertraline Phenibut Reflex stage 41.5 ± 3.4 146.2 ± 21.4 ## 103.5 ± 17.3 137.8 ± 19.1 114.3 ± 20.9 * 111.4 ± 10.6 * Playback (1x introduction) Latent Diving Period 28.8 ± 3.9 137.7 ± 22.5 ## 90.3 ± 18.8 * 130.7 ± 22.1 105.7 ± 23.7 94.3 ± 7.2
* Playback (10-day introduction) 17.8 ± 2.3 108.3 ± 23.2 ## 73.5 ± 11.5 * 118.3 ± 27.6 76.8 ± 21.6 81.6 ± 6.6 *

Example 4. The effect of 9- [2- (4-isopropylphenoxy) ethyl] adenine on the severity of stress-related morphosomatic changes.

Rats after performing behavioral tests 10 days after stress were euthanized under ether anesthesia, opened, and then the adrenal glands, thymus, spleen and stomach were removed. On an analytical balance, adrenal, thymus, and spleen were weighed to determine the relative weight in mg / 100 g of the animal’s weight. The stomach was opened according to lesser curvature and washed, the number of erosions and ulcers on the mucous membrane was calculated, the degree of its damage was determined according to the following scale:

1 point - single erosion,

2 points - multiple erosion,

3 points - single ulcers,

4 points - single erosion and single ulcers,

5 points - multiple erosion and single ulcers,

6 points - multiple ulcers.

Another group of animals was euthanized directly 30-60 minutes after the last stress.

The results of the assessment of stress-induced morphosomatic disorders in animals with "learned helplessness" are presented in tables 11 and 12.

Chronic stress leading to the development of a state of “learned helplessness” in animals was accompanied by morphosomatic disturbances characteristic of stress. So, in stressed animals of the control group (“Control + stress”), the adrenal gland mass coefficient increased statistically significantly, the thymus and spleen mass coefficient decreased, and the degree of ulcerative lesion of the gastric mucosa increased. The listed changes in stressed animals, the euthanasia of which was carried out immediately after the last stress session, were expressed in a statistically significantly greater degree than in animals after 10 days, which indicates the development of the rehabilitation process in the latter. Compound ВМА-99-82 with prophylactic and to a lesser extent with therapeutic administration statistically significantly reduced the severity of changes in all of these indicators, which indicates the presence of this substance anti-stress activity. Comparison preparations also helped to reduce the severity of stress-induced morphosomatic disturbances in animals, however, they were inferior in effectiveness to the studied substance.

Table 11 The severity of stress-induced adrenal hypertrophy and peptic ulcer of the gastric mucosa in animals with "learned helplessness" on the background of the introduction of BMA-99-82 and comparison drugs Experimental groups Adrenal gland mass coefficient (mg / 100 g mass) M ± m The degree of ulcerative lesions of the gastric mucosa M ± m Intact control 14.5 ± 0.8 0.5 ± 0.2 Control + stress (after 30-60 minutes) 26.0 ± 1.5 ## * 4.2 ± 0.4 ## * Control + stress 20.6 ± 1.8 # 2.8 ± 0.4 ## VMA-99-82 15.3 ± 0.8 * 1.3 ± 0.3 * Imipramine 16.8 ± 1.2 * 1.3 ± 0.3 * Sertraline 16.7 ± 1.5 * 2.2 ± 0.3

Phenibut 15.1 ± 0.6 * 1.0 ± 0.4 * Table 12 The severity of stress-induced involution of lymphoid organs in animals with "learned helplessness" on the background of the introduction of BMA-99-82 and comparison drugs Experimental groups Thymus mass coefficient (mg / 100 g mass) M ± m The spleen mass coefficient (mg / 100 g mass) M ± m Intact control 73.8 ± 6.5 444.3 ± 39.6 Control + stress (after 30-60 minutes) 51.7 ± 4.2 # 263.6 ± 17.8 ## * Control + stress 56.5 ± 3.1 # 322.0 ± 13.8 # VMA-99-82 76.4 ± 6.9 * 367.0 ± 22.0 * Imipramine 67.4 ± 3.3 * 331.6 ± 28.7 Sertraline 73.4 ± 4.4 * 343.3 ± 21.7 Phenibut 70.2 ± 5.9 348 ± 28.1

Example 5. The study of the antidepressant properties of 9- [2- (4-isopropyl-phenoxy) ethyl] adenine in a model of chemically induced reserpine depression.

A single intraperitoneal administration of reserpine at a dose of 4 mg / kg causes a deterioration in the general condition of the animals, which is expressed in the development of distinct vegetative disorders, as well as in a decrease in behavioral activity, lethargy and lethargy. In all animals, after the administration of reserpine, there is a decrease in the number of grooming acts, which is accompanied by a decrease in neatness, the animal's coat looks dirty, especially in the genital area and anus, which is associated with the development of diarrhea and increased urination.

All animals were characterized by oligokinesia, in some rats a periodic generalized finely spreading tremor of the whole body was observed. In addition, in all animals treated with reserpine, sensitivity to sound, tactile, and pain stimuli decreased compared with the group of control intact animals. Thus, when approaching the experimenters, the intact control rats showed concern - they actively moved around the home cage, often showed upright stances and acts of short-term grooming, characterizing the behavior of alertness and anxiety. All of the listed behavioral elements in rats exposed to reserpine were suppressed - the animals were indifferent to handling, when testing pain sensitivity, most rats responded to jerking of the tip of their tail with jerking as opposed to intact controls that showed vocalization and pace. The listed phenomena: lethargy, lethargy, decreased behavioral activity and emotional response, decreased sensitivity to external stimuli (sound, tactile, pain), as well as dyskinesias recorded in animals exposed to reserpine, are a consequence of the neuroleptic action of the latter. In general, the general condition in animals that received a single dose of reserpine at a dose of 4 mg / kg can be assessed as a “moderate severity” condition.

With further observation in animals of all experimental groups, as the duration of the therapeutic administration of antidepressants increases, the general condition progressively worsened. Already from the 4th day of the therapeutic administration of all the studied antidepressants - imipramine (15 mg / kg), amitriptyline (15 mg / kg), fluoxetine (20 mg / kg), sertraline (10 mg / kg), venlafaxine (10 mg / kg ) and paroxetine (10 mg / kg) the condition of the animals could be characterized as severe. In animals of all experimental groups lethargy and lethargy increased, the level of response to external stimuli decreased, the symptoms of dyskinesia progressed with the development of parkinsonism syndrome. Dyskinetic manifestations in animals were characterized by the presence of oligokinesia, pro- and retro-pulsive movements. The severity of tremor increased, which by the 7th day of administration in almost all animals of all experimental groups became constant, and in animals treated with imipramine, amitriptyline, fluoxetine and paroxetine (10 mg / kg), it acquired the character of coarse-grained. In addition to the above, in all rats of the experimental groups, muscle rigidity increased, manifested primarily in the “humpiness symptom" (animals acquired a hunched appearance due to rigidity of the muscles of the trunk), which was quantitatively expressed in a decrease in body length from the neck to the base of the tail compared to the intact control rats an average of 3 cm or more. Animals acquired the ability to maintain a pose artificially given to them for a long time, which is a consequence of the development of catalepsy (Table 13). Catalepsy was evaluated in animals on the 7th day of the introduction of substances by a quantitative method: the retention time of the front legs on a 10 cm high bar was recorded with three posing in an artificial pose for 1 min (attempts to give the animal an artificial pose were repeated no more than three times). The cataleptogenic effect of reserpine imipramine was statistically significantly increased, and to a lesser extent paroxetine, fluoxetine, amitriptyline, other comparison drugs showed a tendency to enhance this effect of reserpine. In animals that were administered compound BMA-99-82 at a dose of 30 mg / kg, catalepsy was not detected.

Table 13 The severity of catalepsy in animals with depression caused by the introduction of reserpine against the background of the course of the introduction of BMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + depression Imipramine Paroxetine Fluoxetine Sertraline Venlafaxine Amitriptyline VMA-99-82 Posture Retention Time (s) 9.8 ± 1.5 26.0 ± 3.3 ** 42.8 ± 4.0 *** # 39.5 ± 4.6 *** # 40.2 ± 3.7 *** # 34.2 ± 3.1 *** 29.8 ± 3.6 *** 38.7 ± 4.1 *** # 13.7 ± 1.9

Such an increase in the severity of the antipsychotic effect against the background of the course of therapeutic administration of antidepressants to rats receiving reserpine characterizes the presence of a drug interaction between reserpine and these drugs, which can have both pharmacokinetic and pharmacodynamic character.

Intact control rats throughout the experimental series with modeling of “reserpine depression” maintained a satisfactory condition, normal behavioral activity, and an adequate response to external stimuli. The appearance of the animals of this group was neat, they had a preserved motivation for the behavior of washing and cleaning (grooming).

In animals of the Control + Depression group, which received a single dose of reserpine at a dose of 4 mg / kg, as well as in animals treated with the compound BMA-99-82 within 7 days after administration of reserpine, the state of moderate severity remained throughout the study, and by the 7th day the condition was assessed as satisfactory. They did not show an increase in lethargy, lethargy, dyskinetic phenomena, catalepsy and muscle stiffness were moderate (the “humpiness symptom" in severity corresponded to 1 point and was observed in 4 animals out of 6 in the Control + Depression group and in 2 animals out of 6 in the administration group of compound BMA-99-82). Animals of these groups showed a tendency to reduce these phenomena by the 7th day of the study: the symptoms persisted, but were less pronounced than in the first days of the study.

A similar positive effect of the BMA-99-82 compound was revealed by studying the body weight dynamics of animals with “reserpine depression”, the threshold of their pain sensitivity, the development of pronounced autonomic disorders in animals in the form of blepharoptosis, diarrhea, and increased urination. Thus, the connection ВМА-99-82 helps to reduce the severity of the antipsychotic effect of reserpine, unlike all other studied antidepressants, which have a potentiating effect on this effect of reserpine. The multidirectional effect of the BMA-99-82 compound and the studied antidepressants on the action of reserpine suggests that they have different mechanisms of action.

In the test of forced unavoidable swimming in Porsolt in animals of the Control + Depression group, there was a statistically significant change in the indicators characterizing the severity of depressive behavior in animals (Table 14): a decrease in the latent period of immobilization and an increase in its duration, which persisted throughout the study, which indicates the development of a persistent depressive state in animals exposed to reserpine. The therapeutic administration of the BMA-99-82 compound and all studied antidepressants was accompanied by a statistically significant increase in the latent period of immobilization (i.e., a decrease in the rate of development of a depressive state) and a decrease in its duration (suppression of a state of despair), which indicates that they have an antidepressant effect.

Table 14 The severity of depressive behavior in the Porsolt test in animals with reserpine depression on the background of the introduction of BMA-99-82 and comparison drugs Recorded Indicators (M ± m) Experimental groups Intact control Control + depression Imipramine Paroxetine Fluoxetine Sertraline Venlafaxine Amitriptyline VMA-99-82 1x introduction Latent period of immobilization, s 145.0 ± 8.4 46.5 ± 5.7 85.5 ± 9.0 ** 87.5 ± 7.2 ** 67.3 ± 5.3 * 70.5 ± 6.7 * 97.5 ± 8.2 ** 75.7 ± 6.5 * 74.7 ± 7.1 * Duration of immobilization, s 76.3 ± 6.0 152.3 ± 9.3 87.8 ± 6.4 ** 101.3 ± 6.9 ** 124.5 ± 6.8 * 119.1 ± 9.0 * 93.2 ± 5.7 ** 115.3 ± 6.2 ** 123.5 ± 6.2 * 7 day introduction Latent period of immobilization, s 136.0 ± 9.4 41.2 ± 5.3 70.7 ± 5.8 ** 72.5 ± 7.3 ** 47.5 ± 4.3 76.0 ± 6.9 ** 111.7 ± 8.7 ** 73.0 ± 7.4 ** 68.7 ± 7.1 * Duration of immobilization, s 85.2 ± 4.8 145.2 ± 9.0 108.5 ± 7.9 * 87.7 ± 4.9 ** 110.3 ± 6.0 ** 107.3 ± 9.4 * 73.7 ± 6.7 ** 126.2 ± 7.5 111.7 ± 7.0 *

In addition, in the animals of the Control + Depression group, the Porsolt test showed a statistically significant increase in the behavior of avoiding an aversive situation — the number of jumps and the time of active swimming — which also characterizes the presence of a persistent depression in comparison with the intact control. In rats of all experimental groups that received all the comparison drugs, these indicators statistically significantly increased after a single injection of substances, which also indicates the presence of antidepressant action. At the end of the course therapeutic introduction of all used comparison drugs, the time of active swimming and the number of jumps in animals decreased both in comparison with the intact control and in comparison with the Control + Depression group against the background of a general decrease in locomotor activity associated with the development of antipsychotic syndrome. In animals that were driven by the BMA-99-82 compound, a statistically significant increase in these indicators was observed mainly against a background of single use. After the course application of VMA-99-82, the values of these indicators were comparable with similar indicators of intact control.

When the test was repeated 7 days after the administration of reserpine, the severity of the depressive state in the rats of the Control + Depression group decreased, which may be a consequence of a decrease in the effect of reserpine due to its elimination. It should be noted that when the test was completed at the end of the course administration of the comparison drugs, there was a tendency to a decrease in the severity of the antidepressant effect, which was most observed in fluoxetine, amitriptyline, imipramine and sertraline, to a lesser extent in venlafaxine, paroxetine. The decrease in the effectiveness of the comparison drugs in the Porsolt test after their course use correlated with the severity of the antipsychotic syndrome in animals. Since the studied substance did not potentiate the neuroleptic effect of reserpine, the decrease in the antidepressant effect of BMA-99-82 after its course application may be due to the fact that at the end of the experimental series the severity of reserpine depression decreased.

Example 6. Drug safety study of 9- [2- (4-isopropyl phenoxy) ethyl] adenine.

The study of acute, subacute, chronic and specific toxicity was carried out in accordance with the [Guidelines for the experimental (preclinical) study of new pharmacological substances / Ed. RU. Khabrieva. - M.: Publishing House "Medicine", 2005. - 832 p.].

Acute toxicity experiments were carried out on sexually mature white mice and rats formed in experimental and control groups. Experimental animals 9- [2- (4-isopropylphenoxy) -ethyl] adenine, dissolved in a 50% aqueous solution of dimethyl sulfoxide, was administered in toxic doses once intraperitoneally and intragastrally. Control animals were injected with identical volumes of dimethyl sulfoxide diluted in a ratio of 1: 1 with distilled water. Observation of the animals was carried out for 2 weeks. Based on the results of studies, the LD ₅₀ boundaries were established for the BMA-99-82 compound, which turned out to be equal in mice: with intragastric administration, 992.4 mg / kg in females and 1034.9 mg / kg in males; with intraperitoneal administration, 943.1 mg / kg in females and 962.8 mg / kg in males. In a rat study, the LD ₅₀ of VMA-99-82 for intragastric administration was equal to 4811.7 mg / kg for females and males, and for intraperitoneal administration, it was 3957.9 mg / kg for females and males. Given the results of research and classification of toxicity of substances [Sanotsky I.V. Methods for determination of toxicity and hazard of chemicals (Toxicometry). - M .: "Medicine", 1970. - 343 p.] The connection VMA-99-82 can be attributed to the class of low toxic drugs.

When studying the cumulative properties of 9- [2- (4-isopropyl-phenoxy) ethyl] adenine upon intragastric administration according to the method of Yu.S. Kagan and V.V. Stankevich [Kagan Yu.S. Cumulation. Criteria and methods for its assessment. Prediction of organochlorine intoxications. - In the book: Principles and methods for establishing the maximum permissible concentrations of substances in the air of industrial premises // M .: "Medicine", 1970. - P.49-65] no pronounced cumulation was found in the compound ВМА-99-82.

With a single daily intragastric administration to rats at doses of 10 mg / kg, 75 mg / kg and 150 mg / kg for 1 month, 9- [2- (4-isopropylphenoxy) ethyl] adenine does not have a pathological effect on basic homeostatic constants, that is is safe. When rats were administered to rats at a dose of 300 mg / kg, negative shifts were found (recovering after withdrawal) in general condition, weight gain, peripheral blood, and functional activity of the liver and kidneys. No deaths of animals in this group were observed, pathomorphological studies showed the target-like effect of the substance at a dose of 300 mg / kg on the liver and kidneys. According to the results of chronic studies, it was found that the compound BMA-99-82, when administered intragastrically to rats for 3 months at doses of 10 and 75 mg / kg, does not have a toxic effect on the basic homeostatic constants of animals. Separate electrocardiographic, hematological and biochemical changes detected during the administration of the substance to rats did not go beyond physiological norms. Changes in the behavior of rats treated with the compound at a dose of 150 mg / kg, as well as changes in the content of red blood cells, hemoglobin in the peripheral blood, and impaired functional activity of the liver and kidneys indicate the presence of a negative effect of the substance in this dose on hematopoiesis, detoxification and excretion organs. Based on the studies, it can be concluded that the compound BMA-99-82 with prolonged intragastric administration to rats in doses of 10 to 75 mg / kg does not have a pathological effect on basic homeostatic constants, that is, it is safe.

When studying reproductive toxicity, it was found that 9- [2- (4-isopropylphenoxy) ethyl] adenine, given a 2-month course of intragastric administration to rats in females and males at doses of 10 and 150 mg / kg, does not adversely affect their reproductive function. It was found that under the action of the compound at a dose of 10 mg / kg in males and females, sexual behavior is activated, while under the action of a substance at a dose of 150 mg / kg, sexual behavior in males decreases, but does not change in females. Under the influence of the test substance in males, the spermatogenesis index and the fertilizing function of spermatozoa do not change, but the function of the placenta epithelium improves. Compound BMA-99-82 at doses of 10 and 150 mg / kg, after 2-week administration to rats, to females activates proseptive and receptive sexual behavior, does not affect ovulatory cyclicity, promotes activation of the onset of conception, and tends to have a positive effect on embryogenesis. In studies studying the embryo- and fetotoxic effects recorded in the antenatal period of development, the compound BMA-99-82 was administered to pregnant rats from 6 to 16 days of pregnancy intragastrically at doses of 10, 75 and 150 mg / kg; from 1 to 6 days of pregnancy and from 16 to 19 days of pregnancy at a dose of 150 mg / kg. In studies investigating the antenatal damaging effects recorded in the postnatal period of development, the compound BMA-99-82 was administered intragastrically to pregnant rats from the 6th day of pregnancy and before delivery at a dose of 150 mg / kg. According to the results of studies, the absence of embryo- and fetotoxic effects of the substance in the antenatal and postnatal periods of development was established. The VMA-99-82 compound does not alter fetal death, does not inhibit the quality of the antenatal development of embryos, and does not disrupt the physical and sexual development of the offspring of pups recorded in the postnatal period.

9- [2- (4-Isopropylphenoxy) ethyl] adenine at a conventional therapeutic dose of 10 mg / kg equal to more than 1/400 LD ₅₀ when administered intragastrically to rats to females and males, as well as with a ten-fold increase in the therapeutic dose, does not adversely affect the allergic the status of experimental animals both with a single and with a course introduction and does not contribute to the development of allergic and pseudo-allergic reactions.

The immunotoxic effects of 9- [2- (4-isopropylphenoxy) ethyl] adenine were studied. When administered to BALB / c mice intragastrically at doses of 10 mg / kg and 300 mg / kg, the BMA-99-82 compound did not significantly affect the body weight of the animals during the two-week observation period. It was also shown that the test compound did not lead to noticeable shifts in the mass and cellularity of the lymphoid organs, the thymus and spleen, when administered to mice for 14 days. It was also revealed that the VMA-99-82 compound at doses of 10 mg / kg and 300 mg / kg did not significantly affect the humoral immunity of animals: no statistically significant differences were found in hemagglutinin titers in animals of the experimental and control groups. The test compound, when administered at the therapeutic and maximum doses for two weeks, did not significantly affect cellular immunity, as well as the phagocytic activity of peritoneal macrophages and peripheral blood neutrophils of BALB / c mice.

The tests of the ability of 9- [2- (4-isopropylphenoxy) ethyl] adenine to mutagenic effect in the Ames test showed that the compound itself and its metabolites in concentrations from 0.1 μg to 1000 μg per cup do not induce gene mutations in indicator cells strains of Salmonella typhimurium TA 100, TA98 and TA97. A study of the compound BMA-99-82 in a cytogenetic test showed that both in a single (at a dose of 100 mg / kg) and course (at a dose of 10 mg / kg five times and at a dose of 100 mg / kg five times) intragastric administration to mice, it does not cause an increase in the proportion of cells with damaged chromosomes.

When studying the pharmacological kinetics of 9- [2- (4-isopropyl-phenoxy) ethyl] adenine, a method for its quantitative determination was developed, which allows one to determine the compound in biological samples with high sensitivity and selectivity. The sensitivity of the method is 1 μg / ml, the retention time is 7-7.5 minutes, and the average measurement error is less than 15%. The pharmacokinetic parameters of the VMA-99-82 compound when administered intravenously: T _1/2 = 11.02 h, MRT = 9.56 h, AUC = 74.96 μg × h / ml, Cl = 0.667 l / h × kg, Vd = 10.61 l / kg. When administered orally: T _1/2 = 6.106 h, MTT = 8.523 h, AUC = 49.43 μg × h / ml, Cl = 1.011 l / h × kg, Vd = 8.912 l / kg. The absolute bioavailability was 65.9%.

Thus, 9- [2- (4-isopropylphenoxy) ethyl] adenine has a pronounced anti-stress effect, significantly eliminates stress-induced psycho-emotional, cognitive and morphosomatic disorders. In a whole series of tests, the BMA-99-82 compound is superior in comparison with antidepressant action to comparison drugs: antidepressants imipramine and sertraline, anxiolytic with nootropic effect, phenibut, and also has independent antidepressant activity. There is reason to believe that drugs with this spectrum of psychotropic action will find wide application in clinical practice.

Claims (1)

A drug with antidepressant and antistress action, representing 9- [2- (4-isopropylphenoxy) ethyl] adenine of the formula