Antidiabetic Effect of a New Original NT-3 Dipeptide Mimetic.

It was previously established that the original dipeptide mimetic of the 4th loop of NT-3, hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301), has a pronounced neuroprotective effect in vitro at concentrations of 10-5-10-12 М. In the present study, experiments on the streptozotocin-induced diabetes model in C57Bl/6 mice showed that GTS-301, when administered intraperitoneally for 32 days at doses of 0.1 and 0.5 mg/kg, has antidiabetic activity manifested in a reduction of hyperglycemia and polydipsia and in an increase in animal survival. The results obtained confirm the concept of the similarity of neurochemical mechanisms underlying the regulation of functions of neurons and ß-cells.

Pharmacogenetic Analysis of the Interaction of the Low-Molecular-Weight BDNF Mimetic Dipeptide GSB-106 with TRK Receptors.

Using TrkA or TrkB receptor gene knockout HT-22 cells, the selectivity of the interaction of the low-molecular-weight dipeptide BDNF mimetic GSB-106 (hexamethylenediamide bis(N-monosuccinyl-L-seryl-L-lysine)) with TrkB receptors was shown.

Specifics of Experimental Modeling 8-OH-DPAT-Induced Perseverative Behavior in Mice.

The effect of 5-HT1A receptor agonist 8-OH-DPAT (intraperitoneal injection in doses of 1, 2, and 4 mg/kg) on spontaneous alternation behavior of mice in Y-maze was studied without and with habituation procedure and food reward. In the first case, 8-OH-DPAT administration led to a decrease in spontaneous alternation and locomotor activity in mice. At the same time, 8-OH-DPAT treatment after habituation and food deprivation increased repeated choices of goal arms without affecting locomotor activity, which was consistent with perseverative behavior. 8-OH-DPAT-induced decrease in spontaneous alternation behavior in Y-maze in mice with habituation and food reward is the most suitable procedure for experimental modeling of the perseverative behavior and studying the anticompulsive activity of new substances.

Experimental Study of Antidepressant Properties of Afobazole.

We studied the effects of oral administration of Afobazole in a dose of 10 mg/kg for 5 days on depressive-like behavior of male C57BL/6 mice in the tail suspension test in comparison with amitriptyline (10 mg/kg) or fluoxetine (20 mg/kg) treatment. Afobazole produced an antidepressant effect similar to amitriptyline, but inferior to fluoxetine. The σ1 receptor antagonist BD-1047 in a dose of 5 mg/kg blocked the antidepressant effect of Afobazole, which indicates the involvement of σ1 receptors in the antidepressant effect of the drug.

The First Dipeptide Mimetic of Neurotrofin-3: Design and Pharmacological Properties.

The dimeric dipeptide mimetic hexamethylenediamide bis-(N-monosuccinyl-L-asparaginyl-L-asparagine) (GTS-301) was created on the basis of the structure of the exposed region of the neurotrophin-3 4th loop. The new compound, as well as the full-length neurotrophin, activated the TrkC and TrkB receptors. GTS-301 showed neuroprotective activity in experiments on HT-22 mouse hippocampal cells under conditions of oxidative stress and glutamate toxicity at concentrations of 10-12 and 10-8 M, respectively, and antidepressant-like activity in the forced swimming test on mice with 7-day intraperitoneal administration in doses of 10-40 mg/kg.

Neuroprotective Effect of the Neuropeptide Cycloprolylglycine Depends on AMPA- and TrkB-Receptor Activation.

Previously, we have shown that the endogenous neuropeptide cycloprolylglycine (CPG) is the positive modulator of AMPA receptors and revealed the dependence of its anxiolytic and antihypoxic action on BDNF/Trk signaling. In the present work, we for the first time conducted in vitro experiments using the AMPA receptor blockers DNQX and GYKI 52466 and the Trk receptor blocker K252a. It is shown that the neuroprotective effect of CPG depends on the activation of both AMPA and Trk receptors.

A Low-Molecular-Weight BDNF Mimetic, Dipeptide GSB-214, Prevents Memory Impairment in Rat Models of Alzheimer's Disease.

Brain-derived neurotrophic factor (BDNF) is known to be involved in the pathogenesis of Alzheimer's disease (AD). However, the pharmacological use of full-length neurotrophin is limited, because of its macromolecular protein nature. A dimeric dipeptide mimetic of the BDNF loop 1, bis-(N-monosuccinyl-L-methionyl-L-serine) heptamethylene diamide (GSB-214), was designed at the Zakusov Research Institute of Pharmacology. GSB-214 activates TrkB, PI3K/AKT, and PLC-γ1 in vitro. GSB-214 exhibited a neuroprotective activity during middle cerebral artery occlusion in rats when administered intraperitoneally (i.p.) at a dose of 0.1 mg/kg and improved memory in the novel object recognition test (0.1 and 1.0 mg/kg, i.p.). In the present study, we investigated the effects of GSB-214 on memory in the scopolamine- and steptozotocin-induced AD models, with reference to activation of TrkB receptors. AD was modeled in rats using a chronic i.p. scopolamine injection or a single streptozotocin injection into the cerebral ventricles. GSB-214 was administered within 10 days after the exposure to scopolamine at doses of 0.05, 0.1, and 1 mg/kg (i.p.) or within 14 days after the exposure to streptozotocin at a dose of 0.1 mg/kg (i.p.). The effect of the dipeptide was evaluated in the novel object recognition test; K252A, a selective inhibitor of tyrosine kinase receptors, was used to reveal a dependence between the mnemotropic action and Trk receptors. GSB-214 at doses of 0.05 and 0.1 mg/kg statistically significantly prevented scopolamine-induced long-term memory impairment, while not affecting short-term memory. In the streptozotocin-induced model, GSB-214 completely eliminated the impairment of short-term memory. No mnemotropic effect of GSB-214 was registered when Trk receptors were inhibited by K252A.

Dimeric mimetic of BDNF loop 4 promotes survival of serum-deprived cell through TrkB-dependent apoptosis suppression.

Brain-derived neurotrophic factor (BDNF) is involved in the regulation of neuronal cell growth, differentiation, neuroprotection and synaptic plasticity. Although aberrant BDNF/TrkB signaling is implicated in several neurological, neurodegenerative and psychiatric disorders, neurotrophin-based therapy is challenging and is limited by improper pharmacokinetic properties of BDNF. Dimeric dipeptide compound GSB-106 (bis-(N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide) has earlier been designed to mimic the TrkB-interaction 4 loop of BDNF. It displayed protective effect in various cell-damaging models in vitro. Animal studies uncovered antidepressive and neuroprotective properties upon GSB-106 per os administration. Current study shows that GSB-106 acts similarly to BDNF, promoting survival of serum-deprived neuronal-like SH-SY5Y cells. 100 nmol concentration of GSB-106 provided maximum neurotrophic effect, which corresponds to about 37% of the maximum effect provided by BDNF. Protective properties of GSB-106 arise from its ability to counteract cell apoptosis via activation of TrkB-dependent pro-survival mechanisms, including inactivation of pro-apoptotic BAD protein and suppression of caspases 9 and 3/7. Thus, our study has characterized neurotrophic activity of small dimeric compound GSB-106, which mimics certain biological functions of BDNF and neurotrophin-specific protective mechanisms. GSB-106 also displays similarities to some known low weight peptide and non-peptide TrkB ligands.

Assessment of Susceptibility of Outbred Albino Rats to the Formation of Depression-Like State of Learned Helplessness.

Learned helplessness (a model of depression-like state) was developed in rats by exposure to repeated inescapable electric stimulation and evaluated by the absence of attempts to escape when it could be performed. In randomly grouped outbred white rats, 37.5% animals after the above procedure meet the criterion of learned helplessness. On experimental day 14, the latent period and the number of applied electric shocks prior to the first escape to the safe compartment in rats with learned helplessness were significantly higher than in the control, but no significant differences in these parameters were observed on day 21. The Porsolt forced swimming test performed on days 14 and 21 revealed no differences from the control group. After the rats were divided into low- and high-active subgroups according to their open field behavior, 35% rats with learned helplessness were in the low-active subgroup group and 30% rats with learned helplessness were in the high-active subgroup. On day 14, the parameters of learned helplessness significantly surpassed the control levels only in the low-active subgroup. Only in rats with learned helplessness and low activity in the open field, the immobility time in the Porsolt test was longer than in control low-active rats. These findings attest to advisability of preliminary splitting of outbred animals by their open-field behavior into low- and high-active subgroups and the use of only animals for modeling learned helplessness.

Peptide Mimetic of BDNF Loop 4 Blocks Behavioral Signs of Morphine Withdrawal Syndrome and Prevents the Increase in ΔFosB Level in the Striatum of Rats.

Activity of compound GSB-106, a low-molecular mimetic of loop 4 of the brain neurotrophic factor (BDNF), was studied in experimental morphine withdrawal syndrome simulated in outbred rats. Single and subchronic (5 intraperitoneal injections) administration of GSB-106 in a dose of 0.1 mg/kg significantly reduced the total index of morphine withdrawal syndrome by 55.2 and 45.6%, respectively. GSB-106 reduced the severity of some behavioral signs (piloerection, gnashing of teeth, wet-dog shaking, and runaway attempts), but had no effect on mechanical allodynia formed in the rats with dependence. Subchronic treatment with GSB-106 prevented the increase in the content of ΔFosB (product of early response gene) in the striatum induced by morphine withdrawal. The results confirmed the concept on the involvement of neurotrophins, specifically BDNF and its analogs, in the mechanisms associated with the formation of opiate dependence.

The PI3K/Akt Cascade Is Involved in the Antidiabetic Effect of Compound GSB-214, a Low-Molecular-Weight BDNF Mimetic.

In our previous studies on the streptozotocin model of diabetes we hypothesized that activation of the PI3K/Akt signaling pathway is essential for the realization of the antidiabetic effect of low-molecular-weight NGF and BDNF mimetics. Here we analyze the effect of a specific PI3K/Akt pathway inhibitor (LY 294002) on the antidiabetic effect of the BDNF loop 1 mimetic GSB-214. The experiments on C57BL/6 mice with streptozotocin-induced diabetes showed that GSB-214 attenuated the hyperglycemic effect of streptozotocin and prevented weight loss typical of diabetes, while LY 294002 eliminated these effects of GSB-214. These findings clearly demonstrate the involvement of PI3K/Akt pathway in the implementation of the effects of this low-molecular-weight BDNF mimetic.

Analysis of Cytoprotective Properties of Afobazole in Streptozotocin Model of Diabetes.

Previous in vitro and in vivo studies revealed the neuroprotective effect of anxiolytic Afobazole. Based on similarities in the regulation of functions of neurons and ß cells, we studied the effect of Afobazole on streptozotocin (STZ) model of type 2 diabetes in Wistar rats. Immunohistochemical analysis showed that the decrease in the number of ß cells and a violation of their morphological structure caused by STZ were significantly alleviated by Afobazole administration (10 mg/kg orally for 28 days) to diabetic animals. A correlation between morphometric data and blood glucose level was revealed. A possible role of σ1-receptors in the cytoprotective effects of Afobazole in respect to pancreatic ß cells is discussed.

Dipeptide Mimetics of Different NGF and BDNF Loops Activate PLC-γ1.

Previously, we designed and synthesized dipeptide mimetics of individual loops of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF). It was shown that these mimetics activate the corresponding tyrosine kinase (Trk) receptors and have different patterns of activation of the PI3K/AKT and MAPK/ERK postreceptor signaling pathways in vitro. In the present study, it was shown on HT-22 cells that all these compounds activate the phospholipase C-γ1 (PLC-γ1) cascade.

Cognitive Enhancer Noopept Activates Transcription Factor HIF-1.

The effect of noopept (N-phenylacetyl-L-prolyl-glycine ethyl ester) on the DNA-binding activity of HIF-1 in SH-SH5Y cells and the mechanisms of stabilization of this transcription factor were studied in vitro. Noopept was shown to increase both the basal DNA-binding activity of HIF-1 and the activity induced by various hypoxia mimetics. The mechanism of stabilization of the oxygen-sensitive HIF1α subunit by noopept involves the inhibition of HIF-1 prolyl hydroxylase, which is indirectly indicated by the data obtained using the ODD-Luc reporter, and the positive effect on the level of the HIF1α protein. It was revealed that the effect of noopept is accompanied by changes in gene expression, which belong to different metabolic pathways and are controlled by the transcription factor HIF-1.

The Anxiolytic Effect of the Neuropeptide Cycloprolylglycine Is Mediated by AMPA and TrkB Receptors.

Previously we have shown that the neuropeptide cycloprolylglycine is an endogenous positive modulator of AMPA receptors and assumed that the pharmacological effects of CPG are associated with the brain neurotrophic factor. In this paper, we have first demonstrated that DNQX, an inhibitor of AMPA receptors, and K252A, an ihibitor of Trk receptors, prevented the anxiolytic effect of CPG, which confirms the formulated hypothesis.

Effect of Neuropeptide Cyclo-L-Prolylglycine on Cell Proliferative Activity.

Endogenous neuropeptide cyclo-L-prolylglycine possesses mnemotropic and neuroprotective properties, which can result from its positive effect on the level of brain-derived neurotrophic factor and modulation of activity of insulin-like growth factor-1 and AMPA receptors. For detection of possible mitogenic action of cyclo-L-prolylglycine, we analyzed its effect on proliferative activity of HEK293 and SH-SY5Y cells assessed by expression of Ki-67 proliferation marker, cell cycle examination, and incorporation of modified nucleotide analog EdU into DNA. Cyclo-L-prolylglycine did not affect the level of Ki-67 in examined cell lines and distribution of the cells over G1 and G2 phases of the cell cycle, although it insignificantly reduced the percentage of S phase cells, which attested to the absence of intrinsic mitogenic activity of the peptide. At the same time, cyclo-L-prolylglycine reduced the number of the early apoptotic cells, which can be a mechanisms of its protective action.

Behavioral Effects of Dimeric Dipeptide BDNF Mimetic GSB-106 in a Rat Model of Depressive-Like State.

The effects of GSB-106, a low-molecular mimetic of BDNF loop 4, that represents a substituted dimeric dipeptide bis (N-monosuccinyl-L-seryl-L-lysine) hexamethylenediamide, on cognitive and motor impairments in a model of a depressive-like state in rats caused by unavoidable electric foot-shock were studied using active avoidance and open-field tests. GSB-106 (0.5 mg/kg, per os, 10 days) completely restored the number of avoidance reactions that was reduced in rats exposed to foot-shock and the percentage of trained rats in active avoidance training. In the open-field test, the peptide restored reduced horizontal activity and the number of explored holes. Thus, GSB-106 corrected impaired learning and memory, as well as locomotor activity and exploratory behavior in a model of depression in rats.

A Nerve Growth Factor Dipeptide Mimetic Stimulates Neurogenesis and Synaptogenesis in the Hippocampus and Striatum of Adult Rats with Focal Cerebral Ischemia.

The nerve growth factor (NGF) and its mimetics, which have neuroprotective and neuroregenerative properties, are attractive candidates for developing new drugs for brain injury therapy. A dipeptide mimetic of NGF loop 4, bis(N-succinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), developed at the Zakusov Research Institute of Pharmacology, has the NGF-like ability to activate TrkA receptors, but unlike NGF, GK-2 activates mainly the PI3K/AKT pathway associated with neuroprotection and has no effect on the MAPK cascade associated with hyperalgesia, the main side effect of NGF. That GK-2 possesses neuroprotective activity has been observed in various models of cerebral ischemia. GK-2 was found to statistically significantly reduce the cerebral infarct volume in experimental stroke, even at treatment onset 24 h after injury. This suggests that GK-2 possesses neuroregenerative properties, which may be associated with the activation of neurogenesis and/or synaptogenesis. We studied the effect of GK-2 on neurogenesis and synaptogenesis in experimental ischemic stroke caused by transient occlusion of the middle cerebral artery in rats. GK-2 was administered 6 or 24 h after surgery and then once a day for 7 days. One day after the last administration, proliferative activity in the hippocampus and striatum of the affected hemisphere was assessed using Ki67 and synaptogenesis in the striatum was evaluated using synaptophysin and PSD-95. Ki67 immunoreactivity, both in the striatum and in the hippocampus of the ischemic rats, was found to have dropped by approximately 30% compared to that in the sham-operated controls. Synaptic markers - synaptophysin and PSD-95 - were also statistically significantly reduced, by 14 and 29%, respectively. GK-2 in both administration schedules completely restored the level of Ki67 immunoreactivity in the hippocampus and promoted its increase in the striatum. In addition, GK-2 restored the level of the postsynaptic marker PSD-95, with the therapeutic effect amounting to 70% at the start of its administration after 6 h, and promoted restoration of the level of this marker at the start of administration 24 h after an experimental stroke. GK-2 had no effect on the synaptophysin level. These findings suggest that the neurotrophin mimetic GK-2, which mainly activates one of the main Trk receptor signaling pathways PI3K/ AKT, has a stimulating effect on neurogenesis (and, probably, gliogenesis) and synaptogenesis in experimental cerebral ischemia. This effect may explain the protective effect observed at the start of dipeptide administration 24 h after stroke simulation.

Examination of Cardioprotective Effects of Fabomotizole Hydrochloride in Translational Rat Model of Chronic Heart Failure.

A translational rat model of chronic heart failure was employed to examine the cardioprotective effect of fabomotizole hydrochloride. Fabomotizole therapy for 28 days (15 mg/kg/day intraperitoneally) restored inotropic function of the left ventricle and increased ejection fraction from 54±3 to 65±3% (p=0.001). The inotropic function returned to normal against the background of significantly reduced myocardial expression of angiotensin (p=0.01) and glucocorticoid (p=0.03) receptors and significant increased expression of sigma-1 receptors (p=0.04). Inhibition of abnormal expression of angiotensin and glucocorticoid receptors responsible for activation of the pathological cascades underlying the postinfarction remodeling of the left ventricle as well as activation of the expression of cytoprotective sigma-1 receptors are viewed as the key features of the cardioprotective action of fabomotizole hydrochloride.

Afobazole Alleviates Cognitive Rigidity in Experimental Model of Autism Spectrum Disorders.

Afobazole (10 mg/kg) alleviated cognitive rigidity in BALB/c mice, a phenotypic model of autism spectrum disorders. It improved spatial memory and retraining in T-maze with drinking reinforcement and restored the retrieval of acquired skill during reversal learning in Morris water maze.