The pathogenesis of Parkinson's disease (PD) involves abnormalities in the metabolism of catecholamines. The enzyme quinone reductase 2 (NQO2) reduces quinone derivatives of catecholamines, which promotes the formation of reactive oxygen species (ROS), suggesting a role for NQO2 in the development of cellular damage typical of PD. In the present study, we investigated the relationship between 6-hydroxydophamine (6-OHDA) induced cellular damage and NQO2 activity and its levels in SH-SY5Y cell culture to establish an experimental model to evaluate the pharmacological properties of NQO2 inhibitors. Cellular damage was evaluated using the MTT and comet assays. It was shown that oxidative damage of SH-SY5Y cells upon incubation with 6-OHDA for 6, 12 and 24 h was accompanied by an increase in NQO2 activity. The increase in NQO2 protein level in SH-SY5Y cells was observed 24 h after incubation with 6-OHDA at concentrations of 50 and 100 μM. Oxidative damage of SH-SY5Y cells upon 1 h incubation with 6-OHDA is increased in the presence of the selective enzyme co-substrate 1-benzyl-1,4-dihydronicotinamide (BNAH), but is not accompanied by changes in NQO2 activity and protein levels. The data obtained demonstrate the contribution of NQO2 to the cytotoxic mechanism of 6-OHDA action.

The ability of NQO2 to increase the production of free radicals under enhanced generation of quinone derivatives of catecholamines is considered to be a component of neurodegenerative disease pathogenesis. The present study aimed to investigate the neuroprotective mechanisms of original NQO2 inhibitor M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) in a cellular damage model using NQO2 endogenous substrate adrenochrome (125 µM) and co-substrate BNAH (100 µM). The effects of M-11 (10–100 µM) on the reactive oxygen species (ROS) generation, apoptosis and lesion of nuclear DNA were evaluated using flow cytometry and single-cell gel electrophoresis assay (comet assay). Results were compared with S29434, the reference inhibitor of NQO2. It was found that treatment of HT-22 cells with M-11 results in a decline of ROS production triggered by incubation of cells with NQO2 substrate and co-substrate. Pre-incubation of HT-22 cells with compounds M-11 or S29434 results in a decrease of DNA damage and late apoptotic cell percentage reduction. The obtained results provide a rationale for further development of the M-11 compound as a potential neuroprotective agent.

Induction of BDNF-TrkB signaling is associated with the action mechanisms of conventional and fast-acting antidepressants. GSB-106, developed as a small dimeric dipeptide mimetic of BDNF, was previously shown to produce antidepressant-like effects in the mouse Porsolt test, tail suspension test, Nomura water wheel test, in the chronic social defeat stress model and in the inflammation-induced model of depression. In the present study, we evaluated the effect of chronic per os administration of GSB-106 to Balb/c mice under unpredictable chronic mild stress (UCMS). It was observed for the first time that long term GSB-106 treatment (1 mg/kg, 26 days) during ongoing UCMS procedure ameliorated the depressive-like behaviors in mice as indicated by the Porsolt test. In addition, chronic per os administration of GSB-106 resulted in an increase in BDNF levels, which were found to be decreased in the prefrontal cortex and hippocampus of mice after UCMS. Furthermore, prolonged GSB-106 treatment was accompanied by an increase in the content of pTrkB(706/707) in the prefrontal cortex and by a pronounced increase in the level of pTrkB(816) in both studied brain structures of mice subjected to UCMS procedure. In summary, the present data show that chronic GSB-106 treatment produces an antidepressant-like effect in the unpredictable chronic mild stress model, which is likely to be associated with the regulation of the BDNF-TrkB signaling.

Previously, we demonstrated that the immediate administration of multitarget anxiolytic afobazole slows down the progression of neuronal damage in a 6-hydroxidodamine (6-OHDA) model of Parkinson's disease due to the activation of chaperone Sigma1R. The aim of the present study is to evaluate the therapeutic potential of deferred afobazole administration in this model. Male ICR mice received a unilateral 6-OHDA lesion of the striatum. Fourteen days after the surgery, mice were treated with afobazole, selective Sigma1R agonist PRE-084, selective Sigma1R antagonist BD-1047, and a combination of BD-1047 with afobazole or PRE-084 for another 14 days. The deferred administration of afobazole restored the intrastriatal dopamine content in the 6-OHDA-lesioned striatum and facilitated motor behavior in rotarod tests. The action of afobazole accorded with the effect of Sigma1R selective agonist PRE-084 and was blocked by Sigma1R selective antagonist BD-1047. The present study illustrates the Sigma1R-dependent effects of afobazole in a 6-OHDA model of Parkinson's disease and reveals the therapeutic potential of Sigma1R agonists in treatment of the condition.

Abstract—In an experimental model of Parkinson’s disease, the dopamine content in the striatum, the density of TH+ neurons in the substantia nigra, and the motor activity of mice were studied after the administration of afobazole, which interacts with Sigma-1 and MT1 receptors and regulatory sites of MAO-A and quinone reductase 2 (NQO2, MT3 receptor), and its main metabolite M-11, which selectively interacts only with the regulatory site of NQO2. The drugs were administered intraperitoneally for 14 days with the start of the course 30 minutes after unilateral intrastriatal administration of 5 μg of 6‑hydroxydopamine (6‑OHDA). Afobazole and M‑11 showed neuroprotective properties. In all experiments, the effective dose of afobazole (2.5 mg/kg) was significantly lower than M-11 (7.5 mg/kg). The experimental model used shows the contribution of NQO2 and other drug targets to the antiparkinsonian effect of afobazole.

In this study, 5 µg of 6-hydroxydopamine (6-OHDA) were injected unilaterally into the striatum of mice. The mice were then treated intraperitoneally with afobazole at a dose of 2.5 mg/kg for 14 days starting 30 min after the 6-OHDA injection. The content of monoamines was studied in the striatum of the mice. Afobazole prevented a decrease in the dopamine level in the 6-OHDA-damaged striatum of the experimental animals and did not affect the contents of norepinephrine, serotonin, or its metabolites in the striatum of both the control and 6-OHDA-treated mice.

Resume. Objective. Current insufficiency in treatment of Parkinson’s disease determines the search for new pharmacological targets to achieve neuroprotection and reduce the severity of motor impairment. It is known that ligand activation of chaperone sigma-1 (SigmaR1) and inhibition of quinone reductase 2 (NQO2) cause cytoprotection. Anxiolytic drug afobazole (5-Ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihidrochloride) interacts with SigmaR1 and regulatory site of NQO2, inhibiting it. In vivo and in vitro experiments have demonstrated neuroprotective effect of afobazole. However, the effect of afobazole on motor deficit and motion coordination in model of Parkinson’s disease was not studied. Aim. To study the influence of afobazole on latency to fall in rotarod test in mice with induced 6-hydroxydopamine (6-OHDA) Parkinsonism. Methods. Male CD-1 (ICR) mice were tested at accelerated and constant speed rotarod. Afobazole was administered during 14 days (2.5 mg/kg, ip) at course start 30 minutes after unilateral intrastriatal injection of 5 |xg 6-OHDA. Results. Chronic administration of afobazole (2.5 mg/kg) to hemiparkinsonian mice increases their latency to fall 3.9 fold contrary to placebo treated mice in constant speed rotarod. Afobazole negates 6-OHDA in accelerating rotarod increasing latency to fall 1.6 fold as compared to placebo group. Conclusion. Afobazole prevents motor function impairment in hemiparkinsonian mice with 6-OHDA lesion in rotarod test. Observed effect of afobazole can be a result of it neuroprotective properties derived from SigmaR1 and NQO2 interaction.

Parkinson's disease (PD) is a progressive neurodegenerative disease with limited treatment options. Therefore, the identification of therapeutic targets is urgently needed. Previous studies have shown that the ligand activation of the sigma-1 chaperone (Sigma1R) promotes neuroprotection. The multitarget drug afobazole (5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was shown to interact with Sigma1Rs and prevent decreases in striatal dopamine in the 6-hydroxydopamine (6-OHDA)-induced parkinsonism model. The aim of the present study was to elucidate the role of Sigma1Rs in afobazole pharmacological activity. Using ICR mice we found that administration of afobazole (2.5 mg/kg, i.p.) or selective agonist of Sigma1R PRE-084 (1.0 mg/kg, i.p.) over 14 days normalizes motor disfunction and prevents decreases in dopamine in the 6-OHDA-lesioned striatum. Afobazole administration also prevents the loss of TH + neurons in the substantia nigra. The preadministration of selective Sigma1R antagonist BD-1047 (3.0 mg/kg, i.p.) abolishes the activity of either afobazole or PRE-084, as determined using the rotarod test and the analysis of striatal dopamine content. The current study demonstrates the contribution of Sigma1Rs in the neuroprotective effect of afobazole in the 6-OHDA model of Parkinson's disease and defines the therapeutic perspective of Sigma1R agonists in the clinic.

Resume. Objective. Inhibition of quinone reductase 2 (NQO2) is a perspective target to achieve neuroprotective effect. Anxiolytic drug afobazole (5-Ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihidrochloride) and its main metabolite M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) can interact with melatonin dependent regulatory site of NQO2. Previously we have figured that afobazole inhibits NQO2. However, the role of interaction between M-11 and NQO2 is unclear. Aim. To study the effect of M-11 on activity of NQO2. Methods. The influence of M-11 on activity of human recombinant NQO2 (hNQO2) was measured utilizing fluorescent spectroscopy. Results. M-11 inhibits hNQO2 in concentrations of 0.5 and 1.0 mM, decreasing enzymatic reaction velocity on 12 and 24 % respectively. In same concentrations, M-11 is inferior to afobazole. Conclusion. Compound M-11 inhibits NQO2 and can be used to study pharmacological effects of afobazole caused by interaction with regulatory site of enzyme.

Background. Anxiolytic afobazole (5-Ethoxy-2-[2-(morpholino)-ethylthio] benzimidazole dihidrochloride) has ligand properties towards Sigma-1 chaperone (σ1 receptor, Sigma1R; [3H](+)-pentazocine IC50 = 7.1E-6 M) and the melatonin binding site (MT3 receptor; [125I]2-iodomelatonin IC50 = 9.9E-7 M) of N-Ribosyldihydronicotinamide:Quinone Reductase 2 (NQO2) in ligand binding assay. Further studies revealed its cytoprotective and neuroprotective action in various experimental models. Review analysis of scientific publications and our own findings suggest possible dependence of afobazole cytoprotective effects on both Sigma1R and NQO2. Importantly, the afobazole main metabolite M-11 (2-[2-(3-oxomorpholin-4-il)-ethylthio]-5-ethoxybenzimidazole hydrochloride) has significant affinity only to MT3 receptor ([125I]2-iodomelatonin IC50 = 4.0E-7M). The combination of afobazole, its metabolite M-11 and Sigma1R selective ligands became a very convenient tool to test this hypothesis. The aim of this study was to investigate the contribution of Sigma1R and NQO2 in afobazole cytoprotective effects.

Anxiolytic afobazole (5-Ethoxy-2-[2-(morpholino)-ethylthio] benzimidazole dihidrochloride) has pronounced ligand properties toward Sigma-1 receptor (sigma 1 receptor, SigmaR1) and MT3 receptors. Our previous work demonstrated that afobazole possess cytoprotective effect in the in vitro model of menadione genotoxicity (Woods et al. 1997) through interaction with MT3 receptor (Kadnikov et al. 2014). Present study utilized previously described models to address the contribution of SigmaR1 to cytoprotective action of afobazole. The reduction in afobazole cytoprotective effect observed after preincubation of cell suspension with selective SigmaR1 antagonist BD-1047 revealed an important contribution of SigmaR1 in afobazole-mediated effect. We confirmed our observation using selective SigmaR1 agonist PRE-084. We conclude that pronounced cytoprotective effect of afobazole over PRE-084 is likely achieved by additive SigmaR1 and MT3-mediated effects.

Cell damage depending on activity of quinone reductase 2 (MT3 receptor) was simulated in experiments on bone marrow cell suspension and assessed by menadione-induced DNA breaks measured by comet assay. We analyzed the protective effect of afobazole interacting with MT1, MT3, σ1 receptors, and monoamine oxidase A and its main metabolite M11 that specifi cally binds to MT3 receptors. Both compounds reduced the level of menadione-induced DNA damage (afobazole was effective in lower concentrations in comparison with M-11). Conclusion was made on the contribution of MT3 receptors to the protective effect of afobazole, but the observed concentration differences indicate possible contribution of other targets of anxiolytic drug to the protective mechanisms.

Продолжительное использование препаратов бензодиазепинового ряда нередко приводит к формированию лекарственной зависимости. В данной работе изучено влияние афобазола на показатели поведения в тесте приподнятый крестообразный лабиринт (ПКЛ) и содержание биогенных аминов в структурах мозга при моделировании синдрома отмены диазепама (4 мг/кг/день, внутрибрюшинно, 30 сут) у крыс. Через 48 ч после последней инъекции диазепама афобазол в дозе 5,0 мг/кг препятствовал развитию тревожной реакции, восстанавливая поведенческий паттерн в ПКЛ до уровня животных из контрольной группы. Афобазол повышал сниженное в условиях отмены диазепама содержание дофамина (+ 23,8 %, p < 0,05) в стриатуме. Полученные данные дают основания полагать, что афобазол может быть эффективен при купировании индуцированного отменой диазепама анксиогенеза за счет модуляции функциональной активности дофаминергической системы.