Patent Application: US-201615277341-A

Abstract:
the invention relates to medicine , and more particularly to psychopharmacology , and is directed to an agent for treating and preventing autism spectrum disorders ; said agent is glycine immobilized on detonation nanodiamond particles 2 - 10 nm in size , wherein the content of glycine is from 1 to 21 ± 3 wt . %. the described agent improves the outcome of drug therapy and preventive treatment of autism in children and adults and expands the range of the effective and safe psychotropic medications .

Description:
in accordance with the invention , an agent for the treatment and prevention of autism spectrum disorders is provided , wherein said agent is glycine immobilized on detonation nanodiamond particles 2 - 10 nm in size , wherein the content of glycine is from 1 to 21 ± 3 wt . % ( hereinafter referred to as “ almacine ”). in order to evaluate the prospective use of glycine immobilized on detonation nanodiamond particles 2 - 10 nm in size , wherein the content of glycine is from 1 to 21 ± 3 wt . % ( almacine ), as an agent for the treatment and prevention of autism spectrum disorders , the specific psychopharmacological effect of glycine was compared to that of the pharmaceutical grade glycine and the reference atypical neuroleptic triftazin ( comparator drug ). specific psychopharmacological effects of nanolycine and comparator drugs were studied in the olfactory habituation / dishabituation test simulating impairment of an adequate response to olfactory stimulation , response to the spatial skill acquisition , and the ability to perform new spatial moves after retraining in a water maze , conducted on 330 male balb / c mice , age 5 - 7 weeks , 54 outbred male mice age 2 months , and 18 c57bl / 6 male mice , age 2 - 3 months . the study was conducted according to methods [ 20 , 7 ], respectively . statistical data from the experiment was processed with “ statistica v6 . 0 ” software using the one - way anova test , nonparametric test for variables ( mann - whitney u test ), and paired student &# 39 ; s t - test for intra - group comparison . 1 ) a single administration of almacine at a 1 - mg / kg dose resulted in faster adaptation of the mice to repeated olfactory stimuli and improved the new social odor recognition . nanolycine at a 10 - mg / kg dose also resulted in faster adaptation of the animals to the olfactory stimuli , but behavior of the animals receiving nanolycine in response to new odors did not differ from the behavior of the control animals . the comparator drug triftazin ( 0 . 5 mg / kg ) also improved the new order recognition and accelerated adaptation thereto . the efficacy of nanolycine at both doses was no worse than that of the comparator drug triftazin . the pharmaceutical grade glycine ( active pharmaceutical ingredient ) at a 10 - mg / kg dose was not very effective in this experiment and was much less effective than 1 - and 10 - mg / kg doses of nanolycine . detonation nanodiamonds did not improve the animals &# 39 ; ability to recognize new odors ; 2 ) almacine at a 10 - mg / kg dose ( administered daily over a 6 - day period ) significantly improved the learning process of mice in a water maze both prior and post spatial reversal , while at a 1 - mg / kg dose , it improved the learning process of mice only on the second day of training . the comparator drugs ( administered daily over a 6 - day period ) triftazin ( 0 . 5 mg / kg ) and pharmaceutical grade glycine ( 10 - mg / kg ) had no effect on the animals &# 39 ; learning process , same as the detonation nanodiamonds . example 1 . comparison study of the effect of nanolycine , glycine , triftazin , and nanodiamond on the characteristics of autism in the olfactory habituation / dishabituation test autism impairs the ability to adequately react to social and nonsocial olfactory stimuli . experimental models of said disorders take into consideration the established fact that all interactions between mice , first and foremost , occur via their olfactory signals . thus , the olfactory habituation / dishabituation in the experiment is examined by repeatedly exposing the mice to nonsocial and social odors in the habituation / dishabituation test [ 5 , 12 , 21 ]. the experiment was conducted on male balb / c mice , 5 - 7 - weeks old , weighing 13 - 15 g . c57bl / 6 mice of the same gender as the tested animals were used as social stimuli . outbred male mice , 2 - 3 month old , weighing 24 - 30 g were used as an additional control . the animals were received from the rams nursery “ stolbovaya ” ( moscow oblast ). the animals were kept in a vivarium in accordance with torder # 708n of the ministry of health and social development of the russian federation , aug . 23 , 2010 “ on approval of good laboratory practice ”. the animals were allowed free access to food and water and were fed a full ration of extruded pelletized feed ( gost feed p50258 - 92 ) and drinking water . the temperature was maintained at 20 - 22 ° c . with the light - dark cycle of 12 hours of light and 12 hours of darkness . the animals were kept in polypropylene cages with zinc / chromium steel grates and dust - free litter of wood shavings , 10 mice per cage ( t / 3c ). the mice were kept in accordance with normative document # 1045 - 73 , 04 . 06 . 1973 “ sanitary regulations for arrangement , equipment , and maintenance of vivariums ” approved by the chief public health official . glycine immobilized on detonation nanodiamond particles 2 - 10 nm in size , wherein the content of glycine was from 1 to 21 ± 3 wt . % ( almacine ) at 1 - and 10 - mg / kg doses ; detonation nanodiamonds in concentrations corresponding to the 10 - mg / kg doses of glycine ; the substances were administered to the mice once , intraperitoneally , at 0 . 1 ml per 10 g body weight , 40 min . prior to the experiment . control balb / c animals and outbred mice were intraperitoneally administered 0 . 1 ml of physiological solution per 10 g body weight once , 40 min . prior to the experiment . the olfactory habituation / dishabituation test was used to determine the animals &# 39 ; ability to respond to social and nonsocial olfactory stimuli . the test was conducted according to method [ 21 ] proposed in 2010 . prior to testing , the mice were placed separately into a cage with clean wood shavings for 30 min . a 15 - cm long cotton swab was moistened with water and extended through the cage cover to the height of 5 cm above the wood shavings on the bottom of the cage . each odor was introduced three times : 3 introductions — nonsocial stimulus : diluted flower smell ( lemon extract diluted in a 1 : 100 ratio ( water ); 3 introductions — social stimulus ( odor from an “ unfamiliar ” murine cage with soiled wood shavings ). for the social stimulus , a cotton swab that had been moistened with water was used to swab the bottom of “ another ” soiled murine cage , zigzagging through the cage to cover every corner and the center of the cage . the “ other ” cage should have housed at least 3 c57bl / 6 mice of the same gender as the tested mice . the wood shavings could not be replaced for at least 3 days . the observation was conducted for a period of 2 min . the following parameters were recorded : number of head ( nose ) lifts and turns toward the swab with an odor located no farther than 2 cm away ; after 2 min ., the cotton swab was replaced with another , which was also introduced for 2 min . behavior of outbred and balb / c mice exposed to olfactory stimuli based on the responses to the olfactory stimuli . the characteristic feature of the intact outbred mice behavior during the entire experiment was their recognition of a new odor , which was revealed in their heightened reaction to the swab with the odor at the first exposure to each olfactory stimulus and habituation to the odor at the repeated exposure . the first testing of the “ lemon ” odor in outbred mice showed a 23 . 4 % increase in the response of mice to the olfactory stimulus as compared to their previous exposure to the neutral “ water ” smell ( third exposure ), which had already become habitual . at the first exposure to the olfactory social stimulus “ c57bl / 6 mice odor ” as compared to the third exposure to the “ lemon ” smell , which at that moment became habitual , the response of the mice to the swab with the odor increased 5 . 5 fold ( table 1 ). the pattern of the murine response to a repeated exposure to an odor is shown in table 1 . the table demonstrates that the number of outbred mice that responded to the olfactory stimulus “ water ” at the second exposure was 24 . 2 % lower than the number of mice at the first exposure . when tested with “ lemon ” and “ c57bl / 6 mice ” odors , the animals &# 39 ; response was reduced more significantly , at 74 . 1 % and 51 . 6 %, respectively . at the second exposure to the “ lemon ” and “ c57bl / 6 mice ” odors , balb / c mice showed less pronounced adaptation to the olfactory stimuli than the outbred mice group . thus , the second exposure to the “ lemon ” and “ c57bl / 6 mice ” odors of balb / c mice showed a reduced response , by 42 . 9 % and 25 . 7 % respectively , which was 1 . 7 and 2 . 0 respectively lower than the outbred mice group ( table 1 ). the ability of balb / c mice to recognize the olfactory stimuli “ water ” and “ c57bl / 6 mice odor ” did not differ from that of the outbred mice , which was confirmed by the absence of difference between the responses to new odors in both control groups . the response to the “ lemon ” odor , however , in balb / c mice was 2 . 1 times lower than that of the outbred animals ( table 1 ). the effect of nanolycine on the behavior of balb / c mice at the exposure to olfactory stimuli based on the response to the olfactory stimuli . balb / c mice that were administered 1 - and 10 - mg / kg doses of nanolycine , developed habituation to the “ water ” and “ c57bl / 6 mice odor ” olfactory stimuli after repeated exposures faster than the balb / c controls ( table 2 ). indeed , in the “ nanolycine 1 - mg / kg ” group , the reduced murine response to the “ water ” and “ c57bl / 6 mice odor ” olfactory stimuli after repeated exposures was more statistically significant than after the first exposure : 32 . 5 % 61 . 2 %, respectively . in the “ nanolycine 10 - mg / kg ” group the reduced murine response to the “ water ” and “ c57bl / 6 mice ” odors was 43 . 4 % and 65 . 4 % ( p & lt ; 0 . 05 ). a reduced murine response after the third exposure to the “ water ” and “ c57bl / 6 mice odor ” olfactory stimuli in the “ nanolycine , 1 - mg / kg ” group followed the trend . in addition , the response of the animals receiving nanolycine at a 10 - mg / kg dose to the third “ lemon ” odor exposure was statistically significant : 68 % lower than their response to the second exposure . furthermore , the ability of the animals receiving nanolycine at a 1 - mg / kg dose , to recognize the “ c57bl / 6 mice odor ” stimulus at the first exposure was 2 . 1 times higher than that of balb / c mice ( p & lt ; 0 . 05 ). nanodiamond did not alter the murine ability to recognize olfactory stimuli as compared to the control . however , the animals receiving nanodiamond developed habituation to the “ water ” and “ c57bl / 6 mice odor ” stimuli faster as compared to the balb / c control group animals ( table 2 ). when comparator drugs glycine at a 10 - mg / kg dose and tritazin , at a 0 . 5 mg / kg dose , were administered , habituation to olfactory stimuli also occurred faster as compared to the control balb / c animals . a reduced response to the second exposure of the animals receiving glycine at a 10 - mg / kg dose to the neutral olfactory stimulus “ water ” was statistically significant ( 39 . 2 %) in comparison to the first exposure . glycine caused significantly faster habituation to each consecutive exposure to the “ c57bl / 6 mice odor ” olfactory stimulus . a response to the third exposure to the “ lemon ” odor in this group was also significantly reduced in comparison to the second exposure . all in all , this group of animals showed more statistically significant ( p & lt ; 0 . 01 ) pronounced response to the recognition of all olfactory stimuli as compared to the control balb / c animal group . the second comparator drug triftazin caused significantly faster habituation to each consecutive exposure to the “ water ” and “ c57bl / 6 mice odor ” olfactory stimuli . furthermore , a response of the mice receiving triftazin to the third exposure to the “ lemon ” odor was significantly ( 9 . 5 times ) lower than their response to the second exposure . although recognition of all olfactory stimuli by all the animals in this group at the first exposure was more pronounced in comparison to the control , it was not statistically significant ( table 2 ). thus , nanolycine at 1 - and 10 - mg / kg doses improves the recognition of new social odors and adaptation to olfactory stimuli in the olfactory habituation / dishabituation test . nanolycine at both doses is as effective as the comparator drug triftazin ( 0 . 5 mg / kg ) and more effective than glycine ( 10 - mg / kg ). detonation nanodiamonds do not improve the murine ability to recognize new odors in comparison to the control . example 2 . comparison study of the effect of nanolycine vs . glycine , triftazin , and nanodiamonds on the autism manifestations in learning and relearning tests following spatial “ reversal ” in the morris water maze obsessive repetitive stereotyped behavior , limited interests and activities are typical characteristics of autism [ 22 , 5 ]. repetitive stereotyped behavior in mice includes rotating motion , turning back , repeated sniffing of the same places or objects , excessive grooming , and excessive purposeless motor activity [ 23 , 8 ]. repetitive obsessive behavior of balb / c mice is revealed in the animals &# 39 ; inability to make a decision and choose the correct spatial move when the platform is relocated during retraining in a water maze [ 8 ]. the experiment was conducted on male balb / c mice , 5 - 7 - weeks old , weighing 13 - 15 g . the source of mice and holding conditions were identical to those in example 1 . in example 2 , the tested substances were used the same way as in example 1 . the substances were administered to the mice at 0 . 1 ml per 10 kg of body weight over 6 days . the control balb / c animals and outbred mice were intraperitoneally administered physiological solution at 0 . 1 ml per 10 kg of body weight 40 min . prior to the experiment . a method developed in 2007 and described in [ 7 ] was used to evaluate the effect of nanolycine on the symptoms of autism spectrum disorders . the effect of the tested compounds on the spatial learning process and the ability to make a new spatial move during retraining in a water maze were examined . the test was conducted in the morris water maze . the mice were first taught the skill of locating a platform in a water maze and replicating this spatial skill . the animals were later confronted with a new location of the platform and thus , spatial “ reversal ” was created . during relearning ( relocation of the platform in the water maze ) the animals had to make a new decision and make a correct spatial move . the morris maze is a large circular pool , 122 cm in diameter , 25 cm deep , filled with water at 25 - 28 ° c . a round platform 12 cm in diameter was placed into the pool . the center of the platform was positioned 30 cm away from the edge of the pool . a platform is located 0 . 5 cm above the water . a mouse is placed on the platform for 20 sec . the mouse is then placed in the water at the opposite end of the pool and allowed 60 sec . to find the platform , climb it , and stay there for 20 sec . the process is repeated by placing the mouse in the water in the location of the pool different from the location of the first attempt . each animal is allowed 4 attempts to find the platform . if the animal is unable to find the platform all by itself in 60 sec ., the researcher helps it to find its way to the platform and climb it . during the next 2 days , the platform is placed 0 . 5 cm below the water level . the animals are allowed 4 attempts per day to find the platform in 60 sec . the time gap between the attempts is 20 sec ., wherein they stay on the platform . every day , before their first attempt , the animals are placed on the platform for 20 sec . the time between the moment the animal is placed in the water and the moment the animal climbs on the platform is recorded , as well as the number of effective attempts to find the platform . the animals are placed in the water in three different locations in the part of the pool opposite the platform . 24 hrs . post the second day of training , the replication of the spatial skill is evaluated : the platform is removed and the animals are placed in the pool for 60 sec . once ; the time length of the animal &# 39 ; s stay in the quadrant wherein the platform had been located during the learning stage is recorded . said time is an indicator of learning efficiency and replication of the spatial skill . the day after replicating the spatial skill , the platform is moved to the area of the pool diagonally opposite its previous location . the platform has to be immersed in the water at the same 0 . 5 cm depth below the water level as in the previous days of training . the training ( 2 days ) and replication procedures are repeated according to the scheme described earlier . effect of nanolycine on learning and replicating the spatial skill in balb / c mice in the morris water maze . in the first day of training in the morris water maze , balb / c mice were reported to show a statistically significant increase in the time of the platform search ( by 24 . 0 %) and a decreased number of the effective attempts ( 3 . 6 times ) as compared to the outbred mice ( table 3 ). balb / c mice receiving nanolycine at 1 - and 10 - mg / kg doses , nanodiamond , glycine , and triftazin , didn &# 39 ; t show any statistically significant difference from the linear mice . the trend was ( p & lt ; 0 . 1 , student &# 39 ; s t - test ) toward reduction in the time searching for the platform in the group of mice receiving nanolycine at a 1 - mg / ml dose as compared to the control balb / c mice ( table 3 ). on the second day of training , the linear mice also showed behavior that was significantly different from the behavior of the outbred mice in the time of searching for the platform ( 72 . 2 % increase ) and in the number of effective attempts ( 6 . 4 times reduction ). the first and second day results within the balb / c group didn &# 39 ; t differ from one another , while the outbred animals found the platform on the second say of training significantly faster ( 28 . 6 %) and made a larger number of effective attempts ( 1 . 8 times ) as compared to the first training day ( table 3 ). the animals receiving nanolycine at a 10 - mg / kg dose spent significantly less time ( 18 . 5 %) on the search for the platform and made significantly larger number of effective attempts ( 3 . 6 times ) as compared to the control group . nanolycine at a 10 - mg / kg dose had a positive effect on the ability of the animals to learn , which was confirmed by a statistically significant decrease in the time spent on the search of the platform ( 15 . 6 %) and an increase in the number of effective attempts ( 80 %) on the second day as compared to the first day of training . the 1 - mg / kg of nanolycine dose significantly ( 4 . 4 times ) increased the number of effective attempts to find the platform in comparison to the control group of linear mice . the reduction in the time that took to find the platform for the mice receiving nanolycine was at the trend level ( p & lt ; 0 . 1 , student &# 39 ; s t - test ) and amounted to 14 %. the comparator drug triftazin did not make any impact on the training of the animals in the water maze in comparison to the control group . however , within the same group , on the second day of training , triftazin caused a statistically significant reduction in the time that took to find the platform ( 12 . 3 %) and an increase in the number of effective attempts ( by 62 . 5 %) in comparison to the first day ( table 3 ). when replicating the spatial skill , the time the linear mice spent in the quadrant , in which the platform was located during training , was significantly ( 2 . 3 times ) shorter as compared to the outbred animals ( table 3 ). the time spent in said quadrant for the animals receiving the substances under study did not show a statistically significant difference in comparison to the linear mice group . effect of nanolycine on the behavior of balb / c mice after spatial reversal ( relearning ) in the morris water maze . the time that took mice to find the platform and the number of effective attempts immediately after “ reversal ” ( relearning process ) in the control balb / c was found to be significantly different from the outbred animal group both in the first and second days of their retraining ( table 4 ). the balb / c groups receiving nanolycine at a 1 - mg / kg dose , nanodiamond , glycine , and triftazin , did not show any statistically significant difference from the control group in both recorded parameters on the first and second day of retraining ( table 4 ). the animals receiving nanolycine at a 1 - mg / kg dose showed a statistically significant improvement in their retraining . indeed , on the first day of retraining , the animals made 2 . 4 times the number of effective attempts to find the platform as compared to the control animals ; and on the second day , the number of effective attempts to find the platform was 80 % higher than that of the control group ( table 4 ). on the second day of retraining , the mice receiving nanolycine at a 10 - mg / kg dose spend significantly less time on the search for the platform and made significantly more effective attempts in comparison to the first day of retraining , which demonstrated the positive effect of the preparation during retraining (“ reversal ”) ( table 4 ) when replicating the skill acquired after spatial “ reversal ”, the control animals were found to spend significantly less time than the outbred mice in the quadrant , in which the platform was located during retraining ( table 4 ). nanolycine at a 1 - mg / kg dose significantly ( 3 . 4 times ) increased the time the animals spent in the “ correct ” quadrant . a statistically significant increase of this parameter was also observed in the group of animals receiving nanodiamond , and reached 90 . 1 %. the comparator drugs triftazin and glycine , similar to nanolycine at a 10 - mg / kg dose , did not make any impact on the replication of the newly acquired skill . thus , nanolycine at a 10 - mg / kg dose significantly improves the learning process of balb / c mice in the morris water maze both before and after spatial reversal . nanolycine at a 1 - mg / kg dose improved the learning process of balb / c mice on the second day only . the comparator drugs triftazin and glycine did not make any impact on the animals &# 39 ; learning process . none of the investigated preparations made any impact on the replication of the spatial skill . however , when replicating the spatial skill after “ reversal ” of spatial memory , nanolycine at a 1 - mg / kg dose significantly increased the time balb / c mice spent in the platform quadrant . the experimental results obtained in examples 1 and 2 demonstrated that a single administration of the agent of the present invention at 1 - and 10 - mg / kg doses had a pronounced effect on the social odor recognition and accelerated adaptation to olfactory stimuli in the olfactory habituation / dishabituation test . at both doses , the claimed agent was as effective as the comparator drug triftazin ( 0 . 5 mg / kg ) and more effective than glycine ( 10 - mg / kg ). detonation nanodiamond did not improve recognition of new odors . the claimed agent at a 10 - mg / kg dose ( daily administration over a 6 - day period ) significantly improved the learning process of balb / c mice in the morris water maze both prior and post spatial reversal . the comparator drugs triftazin ( 0 . 5 mg / kg ) and glycine ( 10 - mg / kg ) and nanodiamonds ( administered daily over a 6 - day period ) did not make any impact on the learning process of the animals . following reversal of spatial memory , the claimed agent at a 1 - mg / kg dose ( administered daily over a 6 - day period ) improved replication of the spatial skill and significantly increased the time the animals spend in the platform quadrant . the comparator drugs triftazin ( 0 . 5 mg / kg ) and glycine ( 10 - mg / kg ) as well as nanodiamonds ( administered daily over a 6 - day period ) did not make any impact on the animals &# 39 ; learning process . after reversal of spatial memory , the claimed agent at a 1 - mg / kg dose ( administered daily over a 6 - day period ) improved replication of the spatial skill , significantly increased the time the animals spend in the platform quadrant . the comparator drugs triftazin ( 0 . 5 mg / kg ) and glycine ( 10 - mg / kg ) as well as nanodiamonds ( administered daily over a 6 - day period ) did not make any impact on replication of the spatial skill after reversal of spatial memory . data illustrating anti - autistic activity of glycine , immobilized on detonation nanodiamond particles 2 - 10 nm in size , wherein the content of glycine is 1 wt . %, are shown in table 5 . table 5 demonstrates that increasing the dose of almacin , containing a minimum amount of glycine ( 1 wt . %), results in faster habituation to olfactory stimuli and improved recognition of new social odors , which is expressed in a statistically significant decrease in the experimental animals &# 39 ; ( mice ) response to the first odor exposure . 1 . yakovlev r . yu . nanodiamond - glycine conjugate and method for the preparation thereof . pat . ru 2560700 , 2015 . 2 . rational pharmacotherapy in psychiatric practice : manual for practicing physicians . edited by aleksandrovsky yu . a ., neznanov n . g ., m . littera , 2014 p . 714 - 732 3 . autism spectrum disorders in children . scientific and practical manual . edited by simashkova n . v . m . avtorskaya akademiya , 2013 , 264 p . 4 . manual of clinical psychopharmacology / schatzberg a . f ., cole j . o ., debattista c ., translated from the english , edited by smulevich a . b ., ivanov s . v ., 2 nd edition , m . medpress - inform , 2014 , p . 175 - 220 5 . roullet f . i ., crawley j . n . mouse models of autism : testing hypotheses about molecular mechanisms / j . j . hagan ( ed .) molecular and functional models in neuropsychiatry . curr . topics in behav . neurosci . 2010 . v . 7 . p . 187 - 212 . 6 . moy s . s ., nadler j . j ., perez a . et al . sociability and preference for social novelty in five inbred strains : an approach to assess autistic - like behavior in mice // genes brain behav . 2004 ; 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