RU2759023C2 - Low molecular weight mimetic of brain neurotrophic factor with analgesic, anxiolytic and antiaddictive effects - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to the use of hexamethylenediamine bis-(-N-hexanoyl-L-seryl-L-lysine) as an analgesic, anxiolytic and antiaddictive agent.EFFECT: hexamethylenediamine bis-(-N-hexanoyl-L-seryl-L-lysine) affects nociceptive reaction, anxiety behavior and formed alcoholic motivation, increases the threshold of pain response in the “hot plate” test, demonstrates anxiolytic effect.1 cl, 2 tbl, 9 dwg, 6 ex

Description

Scope of invention

The invention relates to medicine, in particular to pharmacology, and concerns the use of bis - (- N-hexanoyl-L-seryl-L-lysine) hexamethylenediamide (compound GTS-201) as an analgesic, anxiolytic and anti-addictive agent.

State of the art

Neurotrophins belong to a family of growth factors that play an important role in the regulation of survival, growth and differentiation of neurons (Davies, AMJ Neurobiol. 1994; 25: 1334-1348; Snider WD Cell. 1994; 77: 627-638), some of which are potent modulators of synaptic transmission (Berninger B. and Poo M. Curr. Opin. Neurobiol. 1996; 6: 324-330; Lewin GR and Barde Y.-A. Ann. Rev. Neurosci. 1996; 19: 289-317). Among the members of the neurotrophin family, there is a brain-derived neurotrophic factor (BDNF), a 12.4 kDa protein that interacts with tropomyosin tyrosine kinase receptor B (TrkB) with subsequent initiation of downstream signaling via mitogen-activated protein kinase (MAPK) (phospholipase Cγ PLCγ) and phosphoinositol 3-kinase (PI3K) (Kaplan DR and Miller FD Curr. Opin. Cell Biol. 1997; 9: 213-221; Huang EJ, Reichardt LF Annu Rev Biochem. 2003; 72: 609-42).

It is believed that BDNF can serve as a mediator, and not just a modulator of synaptic communication (Park H, Poo MM Nat Rev Neurosci. 2013; 14 (1): 7-23). In addition, BDNF and neurotransmitter signaling cascades can act in close relationship to directly influence synaptic plasticity functions (Fig. 1). The main source of BDNF in the brain is the excitatory glutamatergic synapse, the main synapse for synaptic plasticity, learning, and memory. During plasticity-inducing neuronal activity, BDNF and glutamate are released at synapses, with BDNF secreting more slowly than glutamate. BDNF binds to its TrkB receptor to activate modulatory signaling cascades. At presynaptic terminals, BDNF-TrkB enhances neurotransmitter release. On postsynaptic membranes, BDNF / TrkB signaling increases the function or likelihood of opening ionotropic glutamate receptors. BDNF, as a mediator, can directly influence the late effects of synaptic plasticity, for example, on protein synthesis or gene transcription.

Specific interference with BDNF-related signaling is now regarded as the leading strategy for stimulating neuronal plasticity for the treatment of neurological and psychiatric disorders (Nagahara AN, Tuszynski MH Nat Rev Drug Discov. 2011; 10 (3): 209-19).

Analysis of the scientific literature indicates the effect of BDNF on the activity of the nociceptive system at the spinal and supraspinal levels (Malcangio M. and Lessmann V. Trends Pharmacol. Sci. 2003; 24: 116-121; Pezet S. and McMahon SB Annu. Rev. Neurosci. 2006 ; 29: 507-538). The presence of specific antibodies and modern technologies have made it possible to determine the localization and distribution of BDNF and its high-affinity receptor in central and peripheral neurons. The synthesis and subsequent anterograde axonal transport of BDNF and pro-BDNF have been repeatedly recorded in neurons. Thus, central neurons synthesizing and transporting BDNF have been found in the cortex, hippocampus and macula (Altar CA et al. Nature. 1997; 389: 856-860; Conner, JM et al. J. Neurosci. 1997; 17: 2295-2313). Neurons of all three major descending pathways associated with supraspinal modulation of the nociceptive response are involved in BDNF expression (Akbarian S. et al. J. Neurosci. 2002; 22: 4153-4162; Conner, JM et al. J. Neurosci. 1997; 17 : 2295-2313; Madhav TR et al. Brain Res. Mol. Brain Res. 2001; 93: 56-63.). It should be noted that almost all supraspinal regions associated with nociception contain TrkB-expressing neurons, including centers in the thalamus, reticular formation, hypothalamus, parabrachial nucleus (Arancibia S. et al. Neuroscience. 2007; 146: 864-873; Guo W et al. J. Neurosci. 2006; 26: 126-137; Renn CL et al. Neuroreport. 2006; 17: 1175-1179; Silhol M. et al. Neuroscience. 2005; 132: 613-624), and in the integrative centers of the cortex (Bruns MV and Miller MW Exp. Neurol. 2007; 204: 680-692). Founding article by Kafitz et al. (1999) was one of the first to show that BDNF excites neurons in the hippocampus, cortex and cerebellum as rapidly as the neurotransmitter glutamate (Kafitz K.W. et al. Nature. 1999; 401: 918-921). Although BDNF and TrkB have been found in several brain regions involved in nociception, the functional role of neurotrophin in modulating pain at the supraspinal level is still poorly understood.

According to early experimental studies, an analgesic effect was observed with direct injection of BDNF into the midbrain, close to the periaqueductal gray matter (PAG) and dorsal raphe nuclei (DRNi), when neurotrophin caused a decrease in the response to thermal and chemical (formalin) stimuli, which was accompanied by an increase in the level of a number of neurotransmitters, including endogenous opioids, in the brain and spinal cord. Strengthening the activity of the serotonergic system made it possible to put forward an assumption about the effect of BDNF on the synthesis of serotonin (Siuciak JA et al. Brain Res. 1994; 633: 326-330; Siuciak JA et al. Eur. J. Neurosci. 1995; 7: 663-670; Siuciak JA et al J. Neurosci Res 1998; 52: 149-158). Consistent with these results, Frank et al. (1997) showed that continuous infusion of high doses of BDNF at the PAG and DRNi levels produced analgesia, increasing the tail-flick latency of the tail-flick response over 24 hours, with TrkB protein, but not mRNA, decreased at infusion after 6 and 12 days of BDNF administration without weakening the analgesic effect of neurotrophin (Frank L. et al. Exp. Neurol. 1997; 145: 62-70). A pronounced antinociceptive effect of BDNF during thermal stimulation of nociceptors was recorded after a single intracerebral injection of neurotrophin (Cirulli F. et al. Neurosci. Lett. 2000; 287: 207).

The role of endogenous BDNF in shaping the response to acute pain remains controversial. Although some studies have shown altered nociceptive behavior by neutralizing BDNF in the dorsal horn, the high-frequency stimulation pattern required for neurotrophin release contradicts the role of BDNF in shaping the nociceptive response. BDNF, when administered intrathecally, in some cases causes a pronociceptive response, in others - an antinociceptive effect (Table 1).

This discrepancy can be explained by the different BDNF doses and techniques used. BDNF at high doses appears to have a predominantly antinociceptive effect (Groth R. and Aanonsen L. Pain. 2002; 100: 171-181; Guo W. et al. J. Neurosci. 2006; 26: 126-137), through interaction with low affinity sites and / or activation of various intracellular pathways.

In recent years, there has been a growing interest in the role of BDNF signals in the regulation of fear and anxiety, taking into account the importance of associative synaptic learning in the development of anxiety disorders, which occurs during the conditioned reflex formation of an anxiety reaction (Mineka S, Oehlberg K. Acta Psychol (Amst). 2008; 127 (3): 567-80).

BDNF is one of the most promising molecules for understanding the unfavorable synaptic "learning" underlying the development of anxiety disorders (Lonsdorf TV, Kalisch R Transl Psychiatry. 2011; 1 (): e41), as well as an important factor in stress resistance. For example, mice with a variant of the BDNF Val66Met polymorphism, which is associated with hyperreactivity of the hypothalamic-pituitary-adrenal system in humans and found in patients with depression, exhibit vulnerability to stress, as evidenced by increased anxiety and depressive-like behavior in response to repetitive suppressive stress (Yu H. et al., J Neurosci. 2012.32: 4092-4101). Lower BDNF mRNA and protein content were recorded in the hippocampus in stress-sensitive mice compared to stress-resistant animals (Ieraci A. et al. Neural plasticity. 2016.6212983). In a study by Miao Z. et al (2018), it was found that when modeling psychological social stress in pregnant female mice, anxiety-depressive behavior is formed, which correlates with a decrease in BDNF expression in the hippocampus and prefrontal cortex (Miao Z. et al. Mol Neurobiol. 2018 ; 55 (2): 1097-1111). When considering the relationship between a decrease in BDNF function in the hippocampus and an increased susceptibility to an emotional stress response in old age, a group of scientists put forward a hypothesis according to which a decrease in BDNF, along with aging, induces dysfunction of GABAergic neuroplasticity, and the use of exogenous BDNF can reverse anxiety disorders. caused by the natural aging process, restoring GABA-neuroplasticity (Zhu G. et al. Behav Neurosci. 2019; 133 (2): 212-224). Thus, numerous experimental studies indicate that a decrease in BDNF levels provokes the formation of anxious behavior.

One of the mechanisms illustrating the relationship between emotional stress disorders and alcoholism is the modification of synaptic plasticity through neuroadaptation (Fig. 2).

Studies have shown that ethanol exposure and associated withdrawal symptoms can lead to structural and functional changes at the synaptic level in various regions of the brain as well as in neuronal cultures (Carpenter-Hyland, EP, and Chandler, lJ Eur. J. Neurosci., 2006; 24 (12): 3496-3506, Pandey et al., J. Neurosci. 2008; 28 (10): 2589-2600). The decrease in hippocampal neurotrophic activity due to chronic alcohol exposure, first shown by Walker DW et al., (1992), suggested a relationship between the functional activity of BDNF and alcohol in the adult brain. The neurotrophic factor of the brain in the dorsal striatum has been identified as a central mediator of the homeostatic mechanism, which is activated by acute exposure to alcohol (ethanol) and contributes to a decrease in the sensitivity of rodents to alcohol-induced behavior. Thus, ethanol with a single dose and chronic alcohol consumption in moderate doses, insufficient to sustainably increase blood alcohol levels associated with intoxication (> 80 mg / dL) (Dole VP and Gentry RT. Proc Natl Acad Sci USA. 1984; 81 : 3543-3546), increase BDNF in the striatum, which probably leads to the release of BDNF and TrkB-mediated activation of downstream signaling pathways. Experimental evidence demonstrates that BDNF in the dorsolateral striatum "drives" alcohol motivation through the TrkB-mediated activation of the MAPK / ERK1 / 2 signaling pathway, resulting in upregulation of downstream effector proteins such as dynorphin and dopamine D3 receptor to maintain moderate alcohol consumption (Logrip ML et al. FASEB J. 2008; 22: 2393-2404; Jeanblanc J, et al. J Neurosci. 2006; 26: 1457-1464).

In contrast, intense long-term exposure to ethanol leads to a dysregulation of the BDNF-mediated defense mechanism, accompanied by increased ethanol consumption. Thus, in the work of Logrip ML et al. (2009) showed that the BDNF pathway, which is activated after a single dose of ethanol, is disrupted (weakened) by the end of the 6th week of alcoholism and does not restore its protective function after a 2-week period of alcohol deprivation (Logrip ML et al. J Neurochem. 2009 Jun; 109 (5): 1459-68). Congenital low BDNF expression may dispose rats to higher alcohol consumption, as alcohol-preferring P rats show a decrease in the level of BDNF protein in the nucleus accumbens relative to low-drinker NP rats (Yan QS et al. Brain Res. 2005; 1035 (2): 215 -eight). Modulation of alcohol consumption under conditions of increased and decreased BDNF expression revealed the role of BDNF in reducing alcohol motivation. For example, mice expressing half the normal level of BDNF in the brain show increased alcohol consumption with unrestricted free choice between ethanol and water (Hensler JG et al. J Neurochem. 2003; 85: 1139-1147) and after a period of deprivation (McGough NN, et al. J Neurosci. 2004; 24: 10542-10552), as well as increased alcohol-induced sensitization and pronounced site preference associated with ethanol over wild-type mice (McGough NN, et al. J Neurosci. 2004; 24 : 10542-10552).

The results obtained suggest that the stability of disturbed BDNF signaling may be the cause of the regression of addictive behavior, that is, prolonged unrestricted access to alcohol suggests a possible loss of the protective mechanism, and the absence of alcohol-induced BDNF expression in the dorsal striatum, along with a decrease in BDNF in the cortex and hippocampus, contribute to increased alcohol motivation. It cannot be ruled out that, under these conditions, the regulation of the functional activity of BDNF through the intracellular ERK1 / 2 signaling pathway should be taken into account when identifying new candidates for the development of drugs for the treatment of addictive disorders associated with alcohol use.

At the Research Institute of Pharmacology named after V.V. Zakusov designed and synthesized a dimeric dipeptide mimetic of the 2nd loop BDNF hexamethylenediamide bis - (- N-hexanoyl-L-seryl-L-lysine), or compound GTS-201, which showed neuroprotective properties (Gudasheva T.A. et al. Curr . Pharm. Design. 2019; 25: 729-737) and activated the MAPK / ERK signaling pathway when interacting with the TrkB receptor (Logvinov I.O. et al. Experimental and Clinical Pharmacology. 2018; 81 (S): 145-146) in in vitro studies.

In contrast to the dipeptide mimetic of the 4th loop of BDNF (GSB-106), which has antidepressant activity, GTS-201 in the dose range of 0.1-5.0 mg / kg with intraperitoneal injection did not show activity in the Porsolt test, which makes it possible to reveal the antidepressant effect at the screening stage ( Sazonova N.M. et al. Chemical and pharmaceutical journal. 2018; 52 (9): 14-21).

Currently, there is no information on the analgesic, anxiolytic and antiaddictive properties of bis - (- N-hexanoyl-L-seryl-L-lysine) hexamethylenediamide.

The essence of the invention consists in the use of bis - (- N-hexanoyl-L-seryl-L-lysine) hexamethylenediamide as an analgesic, anxiolytic and anti-addictive agent.

The purpose of the invention is the development of new drugs with analgesic, anxiolytic and anti-addictive properties. This goal was achieved by studying the pharmacological effects of GTS-201 in in vivo experiments in the “elevated cruciform maze”, “hot plate”, “ethanol anesthesia” tests, the effect on ethanol-induced changes in motor activity and formed alcohol motivation.

The technical result of the invention was the expansion of the arsenal of drugs that simultaneously possess a unique pharmacological profile, including analgesic, anxiolytic and anti-addictive effects.

Animals. The experiments were carried out on white outbred male CD-1 mice (n = 112) weighing 18-20 g, inbred male mice C57Bl / 6 (n = 105) and BALB / c (n = 14) with body weight 18-22 g and outbred female rats weighing 500-560 g in the active phase of the experiment (n = 81), subjected to alcoholization under conditions of free choice between a 10 vol% ethanol solution and drinking water (Federal State Budgetary Scientific Institution Scientific Center for Biomedical Technologies of the Federal Medical -biological agency ", branch" Stolbovaya "). Mice were kept in 15 individuals in a T / 3 standard cage, rats - in individual T / 3 standard cages in the vivarium of the V.V. Zakusova "(temperature 21-23 ° C, relative air humidity 40-60%) in natural light and free access to water and briquetted feed for 10 days before testing. The organization and implementation of the work was carried out in accordance with the order of the Ministry of Health of Russia No. 199 dated April 01, 2016 "On the approval of the rules of good laboratory practice." The animals were kept in accordance with SP 2.2.1.3218-14 "Sanitary and Epidemiological Requirements for the Design, Equipment and Maintenance of Experimental Biological Clinics (Vivariums)" dated August 29, 2014 No. 51. The experiments were approved by the Commission on Biomedical Ethics of the Federal State Budgetary Scientific Institution "Research Institute of Pharmacology named after V.V. Zakusov ".

Preparations. GTS-201 (a substance synthesized at the V.V. Zakusov Research Institute of Pharmacology), which was dissolved in water for injection at doses of 0.1-5.0 mg / kg, intraperitoneally (ip), was injected at the rate of 0.1 ml / 10 g (for mice) and 0.1 ml / 100 g (for rats) animal weight ... Animals of the control groups received water for injection. Comparative drugs: narcotic analgesic morphine hydrochloride 3.0 mg / kg, subcutaneously (sc), benzodiazepine anxiolytic phenazepam 0.5 mg / kg, intragastric, benzodiazepine anxiolytic diazepam 1.5 mg / kg, i / b, non-selective opioid blocker 1.0 mg / kg nalox , in / b. Diazepam (Sigma) and phenazepam in the form of solid substances were first dissolved in 1-2 K. Tween-80 and then in water for injection. Naloxone (Sigma) was dissolved in water for injection

Methodology

The hot plate test was used to assess the nociceptive response. The latent reaction time (licking a hind paw or jumping) was recorded using an Ugo Basile analgesimeter (Italy). 1-2 h before the start of the experiment, male mice of the C57Bl / 6 line were selected based on the basic reactivity under the conditions of this experimental model, excluding the mice that remained on the plate heated to 55 ± 0.5 ° C for more than 10 s. A latency period of 20 s (maximum exposure time) was regarded as 100% analgesia. The time of the onset of the reaction in mice was recorded 30, 60, 90, and 120 min after a single injection of the studied drugs. The results obtained were expressed as the maximum possible effect (MPE) in%. MVE = (response latency after drug administration minus background response latency) / (maximum exposure time minus background response latency) X 100% (Le Bars D. et al. Pharmacol Rev. 2001; 53 (4): 597-652 ).

To determine anxiolytic activity, a standard test was used to determine the level of anxiety in rodents "elevated cruciform maze" (PCL), which is a platform raised above the floor level with arms, two of which are closed by walls and the other two are open without partitions. The test substances were administered 30 minutes before the start of testing. CD-1 male mice were placed in the central part of the PCL and recorded: 1) the number of exits into the open (OP) and closed arms (ZR), 2) the time spent in the open arms. Exposure time 5 minutes. After the experiment, for each animal, the ratio of the time spent in the open arms was calculated in relation to the total experiment time equal to 300 seconds, in%. An increase in the time spent in the open arms in the absence of changes in motor activity (the total number of entries into the open and closed arms) was considered as a manifestation of an anxiolytic effect (Walf AA, Frye CA. Nat Protoc. 2007; 2 (2): 322-8: Colick L. G. et al. Bulletin of Experimental Biology and Medicine. 2013; 155 (2): 163-166).

Ethanol anesthesia test. Male mice of the CD-1 line were injected with a 25 vol% ethanol solution at a test dose of 5.5 g / kg, i.p., causing the development of alcoholic anesthesia, which lasts for 1-2 hours, which makes it possible to reveal both weakening and enhancement of the effect ethanol (Anokhina I.P., Kolik L.G. In the book: Guidelines for conducting preclinical studies of drugs of the Federal State Budgetary Institution "SCESMP" of the Ministry of Health and Social Development of Russia. Moscow, 2012, pp. 310-333). The mice were placed on a flat horizontal surface and the time of the onset and end of alcoholic sleep was recorded by adopting the “lateral position” and, accordingly, independently steadily emerging from it. The test substance was administered after the test dose of ethanol after 15 minutes. The control group of animals was injected with an i.p. test dose of ethanol and an isomeric amount of the solvent used for the studied substance. Distilled water was used to prepare an ethanol solution; water for injection was used to dissolve the studied compound GTS-201. Each group includes at least 10-12 individuals. This test is sensitive to ambient temperature and the physiological state of animals.

The spontaneous locomotor activity of mice was assessed in an Opto-Varimex 4 actometer (Columbus Instruments, USA) (Besusso D. et al. Nat Commun. 2013; 4: 2031). The installation is a square plexiglass arena with sides 42 × 42 cm and a height of 20 cm.Around the perimeter of the arena there are four pairs of sensitive optoelectronic sensors (λ = 875 nm) at a height of 2 cm from the floor, automatically registering the parameters of the animal's motor activity and transmitting them to the unit control connected to a computer. The horizontal activity of the animals was recorded continuously for 20 minutes. The timing of animal exposure is based on literature data on the effects of ethanol (2 g / kg, 20 vol% solution) (Melon L.C. and Boehm S.L. Alcohol Clin. Exp Res. 2011; 35 (7): 1351-1360). The test substance was injected 30 minutes before ethanol.

Modeling of chronic alcohol intoxication was carried out on female outbred white rats, which were kept in individual cages with a 10% solution of ethanol and drinking water for 24 weeks; the rats showed a pronounced preference for ethanol in terms of the alcohol-deprivation effect (0.99 ± 0.06 g / kg in terms of ethanol) and under conditions of free choice between ethanol and water (5.05 ± 0.04 g / kg in terms of ethanol) (Vetter CS et al. Alcohol Clin Exp Res. 2007 Jul; 31 (7): 1159-68). The test substances were injected 30 minutes before the presentation of a 2-bottle sample in the ADE test. The level of formed alcoholic motivation in female rats was determined 48 h after ethanol withdrawal using the "alcohol-deprivation effect" test, according to the previously described method. In a study on the preference for ethanol under the subchronic action of the 2nd loop mimetic BDNF in alcoholized females with strong alcohol motivation, the test substances were administered once a day for 10 days. (Kolik L.G. et al. Bulletin of Experimental Biology and Medicine. 2014; 157 (1): 61-65; Nadorova A.V. et al. Neurochemistry. 2014; 31 (2): 147-152).

Statistics. The results were processed using one-way analysis of variance (ANOVA), analysis of variance (ANOVA) for repeated measurements, followed by the use of Duncan's test, Student's t-test, T-test for dependent variables. The critical level of significance is α = 0.05.

Example 1. Evaluation of the analgesic effect of GTS-201 with thermal irritation of nociceptors

GTS-201 at doses of 0.1 and 1.0 mg / kg increased the thresholds of nociceptive response from the 30th to 60th minutes of testing (p <0.01 and p <0.05, respectively), at a dose of 5.0 mg / kg, the mimetic of the 2nd loop BDNF increased the latency period of the reaction at the 30th, 60th and 90th minutes after a single injection compared with the control group (p <0.01, p <0.01, p <0.05, respectively), which indicates the analgesic effect of GTS-201. At 120 min of observation, GTS-201 showed no antinociceptive activity in any of the studied doses (Fig. 3A). In terms of the severity of the effect, GTS-201 was comparable to the reference drug morphine at a dose of 3.0 mg / kg (Fig. 3B) 60 and 90 minutes after drug administration in the hot plate test, which allows assessing nociceptive responses at the supraspinal level.

Example 2. Evaluation of the anxiolytic effect of GTS-201 in the "elevated cruciform maze" test

When assessing the anxiolytic activity of the compound GTS-201 in the dose range of 0.1-5.0 mg / kg, it was found that the mimetic of the 2nd loop of BDNF with a single systemic administration 30 minutes before testing in a dose-dependent manner increased the number of outlets into the open arms at an effective dose of 1.0 mg / kg 3.4 times (p <0.05) and the time spent in the open arms (p <0.05), without affecting the general motor activity of CD-1 mice, which indicates the anti-anxiety effect of GTS-201 (Table 2, Fig.4A , 4B). When comparing the obtained data with a reference drug from the group of classical benzodiazepine tranquilizers, it was shown that phenazepam at an anxiolytic dose of 0.5 mg / kg when administered orally 60 min before testing increased the residence time in the open arms by 1.5 times (p <0.05) compared with the corresponding control. group (table 2). Thus, in terms of anxiolytic activity in the PCL test, GTS-201 in the studied dose range is not inferior to the reference drug.

Example 3. Evaluation of the effect of GTS-201 on the duration of ethanol anesthesia

When assessing the effect of GTS-201 on the acute effects of ethanol in the "ethanol anesthesia" test in CD-1 mice, it was found that its effect on the duration of ethanol sleep was absent, in contrast to the benzodiazepine anxiolytic diazepam, which statistically significantly (p <0.05) increased the time spent in the " lateral position "(Fig. 5). The mimetic of the 2nd loop of BDNF in the dose range of 0.1-5.0 mg / kg with a single systemic administration 30 min before ethanol did not change the duration of the lateral position in CD-1 mice, which makes it possible to exclude the interaction of compounds with ethanol at the pharmacodynamic level and to continue the study of pharmacological properties mimetics in models of alcohol dependence (Fig. 5).

Example 4. Evaluation of the effect of GTS-201 on ethanol-induced suppression of motor activity

When assessing the effect of ethanol at a dose of 2.0 g / kg with a single intraperitoneal injection on the motor activity of C57Bl / 6 mice in an actometer within 20 minutes after administration, it was shown that a statistically significant decrease in motor activity occurs from 11 to 14 minutes of observation (Fig. 4). During this time interval, the studied mimetic of the 2nd loop of BDNF (GTS-201) at a dose of 5 mg / kg when administered 30 min before ethanol statistically significantly (p <0.05) prevented ethanol-induced inhibition of locomotor activity in C57Bl / 6 mice (Fig. . 7).

Example 5. Evaluation of the effect of GTS-201 on the alcohol-deprivation effect in rats chronically alcoholized for 24 weeks

When assessing the antiaddictive potential of the compound GTS-201 in female rats with formed alcohol dependence under conditions of voluntary consumption of 10% ethanol solution, it was found that the mimetic of the 2nd loop BDNF (GTS-201) at a dose of 5 mg / kg with a single systemic administration significantly reduced the level of alcoholic motivation during presentation of 10% ethanol solution and drinking water after 48 hours of alcohol deprivation compared with the control group (p <0.01) and when compared with the background value (p <0.05) (Fig. 8). In terms of the severity of the effect, GTS-201 was not inferior to the reference drug naloxone at a dose of 1.0 mg / kg. The data obtained indicate the ability of GTS-201 to reduce the reinforcing effect of ethanol under these conditions of modeling the state of alcohol dependence.

Example 6. Influence of GTS-201 on the formed alcoholic motivation

The mimetic of the 2nd loop BDNF (GTS-201) at an effective dose of 5 mg / kg with subchronic administration significantly reduced the preference for ethanol from the 1st to the 4th day of administration with free access to ethanol solution and water (Fig.9A), in contrast to naloxone, which under these experimental conditions did not exhibit anti-addictive activity, without reducing the preference for ethanol in rats with formed dependence (Fig. 9B). Thus, the data obtained indicate the ability of GTS-201 to weaken the formed alcoholic motivation in rats with a high level of ethanol consumption.

DESCRIPTION OF DRAWINGS

FIG. 1. General scheme of BDNF signaling pathways (with adaptation by Manju Sasi et al., Pflugers Arch. 2017; 469 (5): 593-610).

FIG. 2. Psychiatric model of the relationship between stress, anxiety and alcohol consumption and its modulation by BDNF (adapted from Moonat S., Pandey SC. Alcohol Res. 2012; 34 (4): 495-505).

FIG. 3. Influence of GTS-201 (A) and morphine (B) on the latent period of the reaction of C57Bl / 6 mice in the hot plate test.

3A.

The abscissa shows the development time of the effect (min).

The ordinate is the maximum possible effect (MPE),%.

Experimental groups:

I - "Control" - white rhombus, line of dots;

II - "GTS-201 0.1 mg / kg" - white square, light gray line

III - "GTS-201 1.0 mg / kg" - black triangle, dark gray line

IV - "GTS-201 5.0 mg / kg" - black square, black line

Repeated measures ANOVA: F (9, 105) = 2.1532, p = 0.03118

Duncan's test:

* - p <0.05, ** - p <0.01 - statistically significant in relation to the control for the "GTS-201 0.1" group;

+ - p <0.05, ++ - p <0.01 - statistically significant in relation to the control for the GTS-201 1.0 group;

x-p <0.05, xx-p <0.01, xxx-p <0.001 statistically significant in relation to the control for the 'TTS-201 5.0' group.

3B.

The abscissa shows the development time of the effect (min).

The ordinate is the maximum possible effect (MPE),%.

Experimental groups:

V - "Control" - white rhombus, line of dots;

VI - "Morphine 3.0 mg / kg" - white square, black line

Repeated measures ANOVA: F (3, 54) = 6.9151, p = 0.00050

Duncan's test:

* - p <0.05, ** - p <0.01 - statistically significant in relation to the control.

FIG. 4A. The effect of GTS-201 and phenazepam on the time spent in the open arms in mice in the "PCL" test. M ± SEM.

The abscissa shows the experimental groups.

CD-1 mice: 1 - Control-1, 2 - GTS-201 0.1 mg / kg, 3 - GTS-201 1.0 mg / kg, 4 - GTS-201 5.0 mg / kg;

BALB / c mice: 5 - Control-2, 6 - Phenazepam 0.5 mg / kg.

Y-axis - time spent in open arms, as a percentage of control;

* - p <0.05 in relation to the corresponding control according to the Student's criterion.

FIG. 4B. The effect of GTS-201 and phenazepam on the outlets into the open arms of PCL in mice. M ± SEM.

The abscissa shows the experimental groups.

CD-1 mice: 1 - Control, 2 - GTS-201 0.1 mg / kg, 3 - GTS-201 1.0 mg / kg, 4 - GTS-201 5.0 mg / kg;

BALB / c mice: 5 - Control, 6 - Phenazepam 0.5 mg / kg. Y-axis - outputs into open arms,%;

* - p <0.05 in relation to the corresponding Control according to the Student's criterion.

FIG. 5. GTS-201 at doses of 0.1, 1.0, and 5.0 mg / kg does not affect the duration of ethanol anesthesia (25% solution, 5.5 g / kg) in outbred mice. M ± SEM.

Abscissa - Experimental groups: 1 - Control-1, 2 - GTS-201 0.1 mg / kg, 3 - TTS-201 1.0 mg / kg, 4 - GTS-201 5.0 mg / kg, 5 - "Control-2", 6 - "Diazepam 1.5 mg / kg".

The ordinate is the duration of ethanol anesthesia (min).

* - p <0.05 in relation to the corresponding Control. Student's test.

FIG. 6. Dynamics of changes in the motor activity of C57 B1 / 6 mice with a single injection of ethanol at a dose of 2 g / kg. M ± SEM.

On the abscissa - Time (min);

Y-axis - Distance traveled (cm).

Experimental groups:

I - "Control" - white rhombus, line of dots;

II - "Ethanol 2 g / kg" - black square, black line

* - p <0.05 statistically significant in relation to the corresponding Control point according to the Student's test.

FIG. 7. Influence of GTS-201 (5.0 mg / kg) on the restoration of locomotor activity in C57Bl / 6 mice, reduced under the action of ethanol (2 g / kg) from 10 to 14 minutes. M ± SEM.

On the abscissa - Experimental groups: 1 - "Control", 2 - "GTS-201", 3 - "Ethanol", 4 - "GTS-201 + Ethanol"; Y-axis - Distance traveled (cm). ANOVA F (3, 43) = 3.1154, p = 0.03586

* - p <0.05 - statistically significant in relation to "Control"

x - p <0.05 - statistically significant in relation to the Ethanol group according to Duncan's test.

FIG. 8. Influence of GTS-201 and naloxone with a single administration on the alcohol-deprivation effect in chronically alcoholized rats. M ± SEM.

On the abscissa - Experimental groups: 1 - "Control", 2 - "GTS-201 1.0 mg / kg", 3 - "GTS-201 5.0 mg / kg", 4 - "Naloxone 1.0 mg / kg". Y-axis - Alcohol deprivation effect (ADE), g / kg ADE (before experiment) - white rectangle; ADE (experience) - gray rectangle.

One-way analysis of variance ANOVA F (2, 25) = 4.6500, p = 0.01919

xx -p <0.05 according to Duncan's criterion

* - p <0.05 relative to the corresponding Control according to the T-test for dependent variables.

FIG. 9. Influence of GTS-201 (A) and naloxone (B) with subchronic administration on ethanol preference in chronically alcoholized rats. M ± SEM.

9A.

On the abscissa - Days of the experiment (days);

Y-axis - Ethanol preference (%).

Experimental groups:

I - "Control" - white rhombus, line of dots;

II - "GTS-201 5.0 mg / kg" - black square, black line

Repeated measures ANOVA - F (7, 112) = 2.7095, p = 0.01241

* -p <0.05 with respect to the "background" point of the "GTS-201 5 mg / kg" group according to Duncan's criterion

9B.

On the abscissa - Days of the experiment (days);

Y-axis - Ethanol preference (%).

Experimental groups:

I - "Control" - white rhombus, line of dots;

II - "Naloxone 1.0 mg / kg" - black square, black line

Repeated measures ANOVA - F (7, 105) = 1.0646, p = 0.39150.

Claims (1)

The use of bis - (- N-hexanoyl-L-seryl-L-lysine) hexamethylenediamide as an analgesic, anxiolytic and anti-addictive agent.

Images