T. A. Gudasheva

The effects of piracetam and its novel peptide analogue GVS-111 on neuronal voltage-gated calcium and potassium channels☆

1. With the use of the two-microelectrode voltage-clamp method, three types of voltage-activated ionic currents were examined in isolated neurons of the snail Helix pomatia: high-threshold Ca2+ current (ICa), high-threshold Ca(2+)-dependent K+ current (IK(Ca)) and high-threshold K+ current independent of Ca2+ (IK(V)). 2. The effect of bath application of the nootropics piracetam and a novel piracetam peptide analog, ethyl ester of N-phenyl-acetyl-L-prolyl-glycine (GVS-111), on these three types of voltage-activated ionic currents was studied. 3. In more than half of the tested cells, ICa was resistant to both piracetam and GVS-111. In the rest of the cells, ICa decreased 19 +/- 7% with 2 mM of piracetam and 39 +/- 14% with 2 microM of GVS-111. 4. IK(V) in almost all cells tested was resistant to piracetam at concentrations up to 2 mM. However, IK(V) in two-thirds of the cells was sensitive to GVS-111, being suppressed 49 +/- 18% with 1 microM GVS-111. 5. IK(Ca) appeared to be the most sensitive current of those studied to both piracetam and GVS-111. Piracetam at 1 mM and GVS-111 at 0.1 microM decreased the amplitude of IK(Ca) in most of the cells examined by 49 +/- 19% and 69 +/- 24%, respectively. 6. The results suggest that piracetam and GVS-111 suppression of voltage-activated calcium and potassium currents of the neuronal membrane may regulate (both up and down) Ca2+ influx into neurons.

Anxiolytic Activity of Endogenous Nootropic Dipeptide Cycloprolylglycine in Elevated Plus-Maze Test

Testing in an elevated plus-maze revealed dose-dependent anxiolytic activity of piracetam analog cycloprolylglycine. Intraperitoneal injection of this agent (0.05 mg/kg) 9-fold prolonged the time spent in open arms compared to the control, without affecting the total motor activity. This effect was stereo-selective: D-enantiomer in doses of 0.05 and 0.1 mg/kg was inactive. Therefore, cycloprolylglycine is similar to piracetam in not only nootropic, but also anxiolytic activity. The existence of an endogenous system responsible for the co-regulation of memory and anxiety is hypothesized.

In Vitro Study of Neuroprotective Properties of GK-2, a New Original Nerve Growth Factor Mimetic

New nerve growth factor (NGF) mimetic GK-2, a substituted dimeric dipeptide, in a concentration of up to 10−9 M produced a protective effect on the culture of immortalized mouse hippocampal neurons (line HT-22) after addition of H2O2 and glutamate. GK-2 in a concentration of 10−8 M protected rat PC-12 pheochromocytoma cells from the neurotoxin MPTP. The neuroprotective effect of this peptide on the model of oxidative stress was also observed in the primary culture of embryonic rat hippocampal neurons.

Endogenous dipeptide cycloprolylglycine shows selective anxiolytic activity in animals with manifest fear reaction.

Experiments on two mouse strains with opposite reactions to emotional stress showed selectivity of the anxiolytic effect of endogenous dipeptide cycloprolylglycine. In the open field test cycloprolylglycine (0.01-0.10 mg/kg intraperitoneally) dose-dependently (1.8-2.1-fold) increased motor activity of BALB/c mice with manifest fear reaction and had no effect on C57Bl/6 mice with active behavior. The content of endogenous cycloprolylglycine in mouse brain correlated with the type of emotional stress reaction: its content in the brain of C57Bl/6 mice 1.5 times surpassed that in BALB/c mice. It is concluded that cycloprolylglycine is involved in the endogenous regulation of fear reaction.

Dimeric dipeptide mimetics of the nerve growth factor Loop 4 and Loop 1 activate TRKA with different patterns of intracellular signal transduction

BackgroundThis study aimed at developing nerve growth factor (NGF) mimetics that selectively activate specific biological signals and, as a result, lack the side effects of the full-length protein. Two dimeric dipeptides, bis-(N-aminocaproyl-glycyl-L-lysine) hexamethylenediamide (GK-6) and bis(N-succinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), were designed based on the most exposed outside fragments of NGF, namely, the loop 1 and loop 4 β-turn sequences, respectively. These dipeptides exhibited neuroprotective activity in vitro at micro-nanomolar concentrations.ResultsStudies on the mechanism of action revealed that both compounds elevate the level of tyrosine kinase A (TrkA) receptor phosphorylation and that they each have different postreceptor signaling patterns. GK-6 increases the levels of extracellular signal-regulated kinase (ERK) and AKT kinase phosphorylation, whereas GK-2 only increases the level of AKT phosphorylation. Apart from the neuroprotective activity, GK-6 promoted differentiation in PC12 cells, whereas GK-2 did not. Furthermore, it was established that the neuroprotective activity of GK-2 was completely abolished by a selective inhibitor of phosphatidylinositol 3-kinase (LY294002) but not by a specific inhibitor of mitogen-activated protein kinases MEK1 and MEK2 (PD98059). In vivo experiments demonstrated that GK-2 did not induce hyperalgesia, which is one of the primary adverse effects of NGF. By contrast, GK-6 produced a significant decrease in the pain threshold of rats as determined by the tail flick test.ConclusionThe data obtained suggest that dimeric dipeptide NGF mimetics are promising candidates in the development of pharmacological agents with NGF-like activity that are free of the main side effect of NGF.

Noopept stimulates the expression of NGF and BDNF in rat hippocampus.

We studied the effect of original dipeptide preparation Noopept (N-phenylacetyl-L-prolylglycine ethyl ester, GVS-111) with nootropic and neuroprotective properties on the expression of mRNA for neurotropic factors NGF and BDNF in rat hippocampus. Expression of NGF and BDNF mRNA in the cerebral cortex and hippocampus was studied by Northern blot analysis. Taking into account the fact that pharmacological activity of Noopept is realized after both acute and chronic treatment, we studied the effect of single and long-term treatment (28 days) with this drug. Expression of the studied neurotropic factors in the cerebral cortex was below the control after single administration of Noopept, while chronic administration caused a slight increase in BDNF expression. In the hippocampus, expression of mRNA for both neurotrophins increased after acute administration of Noopept. Chronic treatment with Noopept was not followed by the development of tolerance, but even potentiated the neurotrophic effect. These changes probably play a role in neuronal restoration. We showed that the nootropic drug increases expression of neurotrophic factors in the hippocampus. Our results are consistent with the hypothesis that neurotrophin synthesis in the hippocampus determines cognitive function, particularly in consolidation and delayed memory retrieval. Published data show that neurotrophic factor deficiency in the hippocampus is observed not only in advanced Alzheimers disease, but also at the stage of mild cognitive impairment (predisease state). In light of this our findings suggest that Noopept holds much promise to prevent the development of Alzheimers disease in patients with mild cognitive impairment. Moreover, therapeutic effectiveness of Noopept should be evaluated at the initial stage of Alzheimers disease.

Pharmacogenetic Study of Anxiolytic Effects of New Cholecystokinin Receptor Antagonists in Animals with Different Levels of Emotionality

Effects of two new peptide antagonists of central cholecystokinin receptors, GB-101 (0.05-0.40 mg/kg) and GB-115 (0.006-0.100 mg/kg), on the behavior of inbred animals differing by the reactions to emotional stress were studied in standard tests for evaluation of tranquilizers. The test drugs dose-dependently increased the total locomotor activity in the open field test in Balb/c mice and had no effect on the behavior of C57Bl/6 mice. GB-101 (0.4 mg/kg) and GB-115 (0.025 and 0.05 mg/kg) produced an anxiolytic effect on MR rats (but not on MNRA rats) in the elevated plus-maze and conflict tests. These data confirm anxioselective effects of the test compounds.

Mimetics of brain-derived neurotrophic factor loops 1 and 4 are active in a model of ischemic stroke in rats

Background Two dimeric dipeptides, bis-(N-monosuccinyl-l-seryl-l-lysine)hexamethylenediamide (GSB-106) and bis-(N-monosuccinyl-l-methionyl-l-serine) heptamethylenediamide (GSB-214), were designed based on the brain-derived neurotrophic factor (BDNF) loop 4 and loop 1 β-turn sequences, respectively. Earlier, both of these dipeptides were shown to exhibit neuroprotective activity in vitro (10−5–10−8 M). The present study aimed to investigate the mechanisms of action of these peptides and their neuroprotective activity in an experimental stroke model. Methods We used western blot and HT-22 hippocampal neuronal cell line to investigate whether these peptides induced phosphorylation of the TrkB receptor and the AKT and ERK kinases. Rat middle cerebral artery occlusion (MCAO) was used as a stroke model. GSB-106 and GSB-214 were administered intraperitoneally (0.1 mg (1.3×10−7 mol)/kg) 4 hours after MCAO and daily for 7 days. The cerebral infarct volumes were measured with 2,3,5-triphenyltetrazolium chloride staining 21 days after MCAO. Results Both compounds were shown to elevate the TrkB phosphorylation level while having different post-receptor signaling patterns. GSB-106 activated the PI3K/AKT and MAPK/ERK pathways simultaneously, whereas GSB-214 activated the PI3K/AKT only. In experimental stroke, the reduction of cerebral infarct volume by GSB-106 (∼66%) was significantly greater than that of GSB-214 (∼28% reduction), which could be explained by the fundamental role of the MAPK/ERK pathway in neurogenesis and neuroplasticity. Notably, between these two dipeptides, only GSB-106 exhibited antidepressant activity, as was found previously. Conclusion The results provided support for the beneficial pharmacological properties of BDNF loop 4 mimetic GSB-106, thereby suggesting a potential role for this dipeptide as a therapeutic agent.

Regional and Subcellular Localization of Cycloprolylglycine in Rat Brain

HPLC analysis showed that endogenous cyclopeptide cycloprolylglycine exhibiting mnemotropic and anxiolytic properties is nonuniformly distributed between brain structures in rats: its contents in the whole brain, cortex, and hippocampus were 29.2±1.6, 38.9±8.0, 126.4±32.4 nmol/g, respectively. Cycloprolylglycine distribution between subcellular fractions of brain neurons is also nonuniform: 60% cyclopeptide appeared in the nuclear fraction.

Behavioral effect of dipeptide NGF mimetic GK-2 in an in vivo model of rat traumatic brain injury and its neuroprotective and regenerative properties in vitro

A protective behavioral effect of a nerve growth factor dipeptide mimetic GK-2 in the model of open focal trauma of rat brain sensorimotor cortex and its antioxidative and regenerative properties in cultures of rat cerebellar granule cells and mouse embryonal spinal ganglion, respectively, were studied. Intraperitoneal injections of GK-2 (1 mg/kg) for 5 days daily after traumatic brain injury improved significantly motor function of limbs. Moreover, supplementation the incubation medium with GK-2 (0.5–1.5 mg/l) decreased neuronal death induced by H2O2 in cerebellar granule cell cultures and stimulated neurite outgrowth from cultured mouse embryonal spinal ganglia. Our results suggest that GK-2 exhibits pronounced positive behavioral effect in vivo as well as neuroprotective and regenerative effects in vitro, and that these neuroprotective properties probably associated with cell survival but not with cell differentiation pathway.