Research in the Field of Drug Design and Development.

The processes used by academic and industrial scientists to discover new drugs have recently experienced a true renaissance, with many new and exciting techniques being developed over the past 5-10 years alone. Drug design and discovery, and the search for new safe and well-tolerated compounds, as well as the ineffectiveness of existing therapies, and society's insufficient knowledge concerning the prophylactics and pharmacotherapy of the most common diseases today, comprise a serious challenge. This can influence not only the quality of human life, but also the health of whole societies, which became evident during the COVID-19 pandemic. In general, the process of drug development consists of three main stages: drug discovery, preclinical development using cell-based and animal models/tests, clinical trials on humans and, finally, forward moving toward the step of obtaining regulatory approval, in order to market the potential drug. In this review, we will attempt to outline the first three most important consecutive phases in drug design and development, based on the experience of three cooperating and complementary academic centers of the Visegrád group; i.e., Medical University of Lublin, Poland, Masaryk University of Brno, Czech Republic, and Comenius University Bratislava, Slovak Republic.

Development and Characterization of Novel Selective, Non-Basic Dopamine D2 Receptor Antagonists for the Treatment of Schizophrenia.

The dopamine D2 receptor, which belongs to the family of G protein-coupled receptors (GPCR), is an important and well-validated drug target in the field of medicinal chemistry due to its wide distribution, particularly in the central nervous system, and involvement in the pathomechanism of many disorders thereof. Schizophrenia is one of the most frequent diseases associated with disorders in dopaminergic neurotransmission, and in which the D2 receptor is the main target for the drugs used. In this work, we aimed at discovering new selective D2 receptor antagonists with potential antipsychotic activity. Twenty-three compounds were synthesized, based on the scaffold represented by the D2AAK2 compound, which was discovered by our group. This compound is an interesting example of a D2 receptor ligand because of its non-classical binding to this target. Radioligand binding assays and SAR analysis indicated structural modifications of D2AAK2 that are possible to maintain its activity. These findings were further rationalized using molecular modeling. Three active derivatives were identified as D2 receptor antagonists in cAMP signaling assays, and the selected most active compound 17 was subjected to X-ray studies to investigate its stable conformation in the solid state. Finally, effects of 17 assessed in animal models confirmed its antipsychotic activity in vivo.

Impact of Mephedrone on Fear Memory in Adolescent Rats: Involvement of Matrix Metalloproteinase-9 (MMP-9) and N-Methyl-D-aspartate (NMDA) Receptor.

Treatment of Post-Traumatic Stress Disorder (PTSD) is complicated by the presence of drug use disorder comorbidity. Here, we examine whether conditioned fear (PTSD model) modifies the rewarding effect of mephedrone and if repeated mephedrone injections have impact on trauma-related behaviors (fear sensitization, extinction, and recall of the fear reaction). We also analyzed whether these trauma-induced changes were associated with exacerbation in metalloproteinase-9 (MMP-9) and the GluN2A and GluN2B subunits of N-methyl-D-aspartate (NMDA) glutamate receptor expression in such brain structures as the hippocampus and basolateral amygdala. Male adolescent rats underwent trauma exposure (1.5 mA footshock), followed 7 days later by a conditioned place preference training with mephedrone. Next, the post-conditioning test was performed. Fear sensitization, conditioned fear, anxiety-like behavior, extinction acquisition and relapse were then assessed to evaluate behavioral changes. MMP-9, GluN2A and GluN2B were subsequently measured. Trauma-exposed rats subjected to mephedrone treatment acquired a strong place preference and exhibited impairment in fear extinction and reinstatement. Mephedrone had no effect on trauma-induced MMP-9 level in the basolateral amygdala, but decreased it in the hippocampus. GluN2B expression was decreased in the hippocampus, but increased in the basolateral amygdala of mephedrone-treated stressed rats. These data suggest that the modification of the hippocampus and basolateral amygdala due to mephedrone use can induce fear memory impairment and drug seeking behavior in adolescent male rats.

Synthesis, Structural and Behavioral Studies of Indole Derivatives D2AAK5, D2AAK6 and D2AAK7 as Serotonin 5-HT1A and 5-HT2A Receptor Ligands.

Serotonin receptors are involved in a number of physiological functions and regulate aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, sleep, and thermoregulation. Here we report synthesis and detailed structural and behavioral studies of three indole derivatives: D2AAK5, D2AAK6, and D2AAK7 as serotonin 5-HT1A and 5-HT2A receptor ligands. X-ray studies revealed that the D2AAK5 compound crystallizes in centrosymmetric triclinic space group with one molecule in the asymmetric unit. The main interaction between the ligands and the receptors is the salt bridge between the protonatable nitrogen atom of the ligands and the conserved Asp (3.32) of the receptors. The complexes were stable in the molecular dynamic simulations. MD revealed that the studied ligands are relatively stable in their binding sites, with the exception of D2AAK7 in the serotonin 5-HT1A receptor. D2AAK7 exerts anxiolytic activity in the EPM test, while D2AAK5 has a beneficial effect on the memory processes in the PA test.

Funnel metadynamics and behavioral studies reveal complex effect of D2AAK1 ligand on anxiety-like processes.

Anxiety is a troublesome symptom for many patients, especially those suffering from schizophrenia. Its regulation involves serotonin receptors, targeted e.g. by antipsychotics or psychedelics such as LSD. 5-HT2A receptors are known for an extremely long LSD residence time, enabling minute doses to exert a long-lasting effect. In this work, we explore the changes in anxiety-like processes induced by the previously reported antipsychotic, D2AAK1. In vivo studies revealed that the effect of D2AAK1 on the anxiety is mediated through serotonin 5-HT1A and 5-HT2A receptors, and that it is time-dependent (anxiogenic after 30 min, anxiolytic after 60 min) and dose-dependent. The funnel metadynamics simulations suggest complicated ligand-5HT2AR interactions, involving an allosteric site located under the third extracellular loop, which is a possible explanation of the time-dependency. The binding of D2AAK1 at the allosteric site results in a broader opening of the extracellular receptor entry, possibly altering the binding kinetics of orthosteric ligands.

Synthesis, Docking Studies and Pharmacological Evaluation of Serotoninergic Ligands Containing a 5-Norbornene-2-Carboxamide Nucleus.

A new series of 5-norbornene-2-carboxamide derivatives was prepared and their affinities to the 5-HT1A, 5-HT2A, and 5-HT2C receptors were evaluated and compared to a previously synthesized series of derivatives characterized by exo-N-hydroxy-5-norbornene-2,3-dicarboximidenucleus, in order to identify selective ligands for the above-mentioned subtype receptors. Arylpiperazines represents one of the most important classes of 5-HT1AR ligands, and recent research concerning new derivatives has been focused on the modification of one or more portions of such pharmacophore. The combination of structural elements (heterocyclic nucleus, propyl chain and 4-substituted piperazine), known to be critical to the affinity to 5-HT1A receptors, and the proper selection of substituents led to compounds with high specificity and affinity towards serotoninergic receptors. The most active compounds were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that Norbo-4 and Norbo-18 were the most active and promising derivatives for the serotonin receptor considered in this study.

Cebranopadol as a Novel Promising Agent for the Treatment of Pain.

Opioids are used to treat pain, but despite their effectiveness, they possess several side effects such as respiratory depression, tolerance and physical dependence. Cebranopadol has been evaluated as a solution to this problem. The compound acts on the mu opioid receptor and the nociceptin/orphanin receptor and these receptors co-activation can reduce opioid side-effects without compromising analgesia. In the present review, we have compiled information on the effects of cebranopadol, its pharmacokinetics, and clinical trials involving cebranopadol, to further explore its promise in pain management.

In vitro and in vivo evaluation of antioxidant and neuroprotective properties of antipsychotic D2AAK1.

The susceptibility of neurons to free radical toxicity partially underlies the pathomechanism of neurodegenerative diseases. On the other hand, excitotoxicity also contributes to neurodegeneration. Our previous studies demonstrated the unique properties of D2AAK1 as a potent multi-target ligand of aminergic G protein-coupled receptors (GPCRs) which dose-dependently stimulates growth, survival of neurons, and promotes their integrity. The aim of our study was to investigate the potential neuroprotective and antioxidant properties of D2AAK1. Here we show that D2AAK1 activates cellular and molecular neuroprotective mechanisms, prevents cells from excitotoxicity and free radicals. Furthermore, D2AAK1 induced no genotoxic events in neuronal cells in vitro. Most importantly, D2AAK1 protects neurons from the effects of high temperatures by molecular chaperones activation. The D2AAK1 effects on selected organs was further evaluated in mice and no pathological changes were observed after chronic administration. In the light of our experiments, D2AAK1 can be further developed into a potential treatment for neurodegenerative diseases, in particular related to memory impairment. In summary, D2AAK1 has promising properties for potential treatments of neurodegenerative diseases.

Alteration of Ethanol Reward by Prior Mephedrone Exposure: The Role of Age and Matrix Metalloproteinase-9 (MMP-9).

Mephedrone, a synthetic cathinone, is widely abused by adolescents and young adults. The aim of this study was to determine: (i) whether prior mephedrone exposure would alter ethanol reward and (ii) whether age and matrix metalloproteinase-9 (MMP-9) are important in this regard. In our research, male Wistar rats at postnatal day 30 (PND30) received mephedrone at the dose of 10 mg/kg, i.p., 3 times a day for 7 days. To clarify the role of MMP-9 in the mephedrone effects, one mephedrone-treated group received minocycline, as an MMP-9 antagonist. Animals were then assigned to conditioned place preference (CPP) procedure at PND38 (adolescent) or at PND69 (adult). After the CPP test (PND48/79), expression of dopamine D1 receptors (D1R), Cav1.2 (a subtype of L-type calcium channels), and MMP-9 was quantified in the rat ventral striatum (vSTR). The influence of mephedrone administration on the N-methyl-D-aspartate glutamate receptors (NMDAR) subunits (GluN1, GluN2A, and GluN2B) was then assessed in the vSTR of adult rats (only). These results indicate that, in contrast with adolescent rats, adult rats with prior mephedrone administration appear to be more sensitive to the ethanol effect in the CPP test under the drug-free state. The mephedrone effect in adult rats was associated with upregulation of D1R, NMDAR/GluN2B, MMP-9, and Cav1.2 signaling. MMP-9 appears to contribute to these changes in proteins expression because minocycline pretreatment blocked mephedrone-evoked sensitivity to ethanol reward. Thus, our results suggest that prior mephedrone exposure differentially alters ethanol reward in adolescent and adult rats.

Rapamycin Improves Spatial Learning Deficits, Vulnerability to Alcohol Addiction and Altered Expression of the GluN2B Subunit of the NMDA Receptor in Adult Rats Exposed to Ethanol during the Neonatal Period.

Ethanol exposure during pregnancy alters the mammalian target of rapamycin (mTOR) signaling pathway in the fetal brain. Hence, in adult rats exposed to ethanol during the neonatal period, we investigated the influence of rapamycin, an mTOR Complex 1 (mTORC1) inhibitor, on deficits in spatial memory and reversal learning in the Barnes maze task, as well as the ethanol-induced rewarding effects (1.0 or 1.5 g/kg) using the conditioning place preference (CPP) paradigm. Rapamycin (3 and 10 mg/kg) was given before intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4-9 (an equivalent to the third trimester of human pregnancy). Spatial memory/reversal learning and rewarding ethanol effect were evaluated in adult (PND60-70) rats. Additionally, the impact of rapamycin pre-treatment on the expression of the GluN2B subunit of NMDA receptor in the brain was assessed in adult rats. Our results show that neonatal ethanol exposure induced deficits in spatial memory and reversal learning in adulthood, but the reversal learning outcome may have been due to spatial learning impairments rather than cognitive flexibility impairments. Furthermore, in adulthood the ethanol treated rats were also more sensitive to the rewarding effect of ethanol than the control group. Rapamycin prevented the neonatal effect of ethanol and normalized the GluN2B down-regulation in the hippocampus and the prefrontal cortex, as well as normalized this subunit's up-regulation in the striatum of adult rats. Our results suggest that rapamycin and related drugs may hold promise as a preventive therapy for fetal alcohol spectrum disorders.

N-(3-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}propyl)-1H-indazole-3-carboxamide (D2AAK3) as a potential antipsychotic: In vitro, in silico and in vivo evaluation of a multi-target ligand.

Schizophrenia is a mental illness of not adequately understood causes that is not satisfactorily enough treated by current antipsychotics. In search for novel potential antipsychotics we performed structure-based virtual screening aimed to identify new dopamine D2 receptor antagonists. We found compound D2AAK3 with affinity to dopamine D2 receptor of 115 nM. D2AAK3 possesses additional nanomolar or low micromolar affinity to D1, D3, 5-HT1A, 5-HT2A and 5-HT7 receptors, which makes it a good hit for further development as a multifunctional ligand. The compound has also some affinity to M1 and H1 receptors. We used homology modeling, molecular docking and molecular dynamics to study interactions of D2AAK3 with its molecular targets at the molecular level. In behavioral studies D2AAK3 decreases amphetamine-induced hyperactivity (when compared to the amphetamine-treated group) measured as spontaneous locomotor activity in mice. In addition, passive avoidance test demonstrated that D2AAK3 improves memory consolidation after acute treatment in mice. Elevated plus maze tests indicated that D2AAK3 induces anxiogenic activity 30 min after acute treatment, whereas this effect has no longer been observed 60 min after administration of the studied compound in mice.

Synthesis, docking studies, and pharmacological evaluation of 2-hydroxypropyl-4-arylpiperazine derivatives as serotoninergic ligands.

A new series of norbornene and exo-N-hydroxy-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboximide derivatives was prepared, and their affinities to the 5-HT1A , 5-HT2A , and 5-HT2C receptors were evaluated and compared with a previously synthesized series of derivatives characterized by the same nuclei, to identify selective ligands for the subtype receptors. Arylpiperazines represent one of the most important classes of 5-HT1A R ligands, and the research of new derivatives has been focused on the modification of one or more portions of this pharmacophore. The combination of structural elements (heterocyclic nucleus, hydroxyalkyl chain, and 4-substituted piperazine), known to be critical for the affinity to 5-HT1A receptors, and the proper selection of substituents resulted in compounds with high specificity and affinity toward serotoninergic receptors. The most active compounds were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that 3e, 4j, and 4n were the most active and promising derivatives for the serotonin receptor considered in this study.

Effects of Mephedrone and Amphetamine Exposure during Adolescence on Spatial Memory in Adulthood: Behavioral and Neurochemical Analysis.

A synthetic cathinone, mephedrone is widely abused by adolescents and young adults. Despite its widespread use, little is known regarding its long-term effects on cognitive function. Therefore, we assessed, for the first time, whether (A) repeated mephedrone (30 mg/kg, i.p., 10 days, once a day) exposure during adolescence (PND 40) induces deleterious effects on spatial memory and reversal learning (Barnes maze task) in adult (PND 71-84) rats and whether (B) these effects were comparable to amphetamine (2.5 mg/kg, i.p.). Furthermore, the influence of these drugs on MMP-9, NMDA receptor subunits (GluN1, GluN2A/2B) and PSD-95 protein expression were assessed in adult rats. The drug effects were evaluated at doses that per se induce rewarding/reinforcing effects in rats. Our results showed deficits in spatial memory (delayed effect of amphetamine) and reversal learning in adult rats that received mephedrone/amphetamine in adolescence. However, the reversal learning impairment may actually have been due to spatial learning rather than cognitive flexibility impairments. Furthermore, mephedrone, but not amphetamine, enhanced with delayed onset, MMP-9 levels in the prefrontal cortex and the hippocampus. Mephedrone given during adolescence induced changes in MMP-9 level and up-regulation of the GluN2B-containing NMDA receptor (prefrontal cortex and hippocampus) in young adult (PND 63) and adult (PND 87) rats. Finally, in adult rats, PSD-95 expression was increased in the prefrontal cortex and decreased in the hippocampus. In contrast, in adult rats exposed to amphetamine in adolescence, GluN2A subunit and PSD-95 expression were decreased (down-regulated) in the hippocampus. Thus, in mephedrone-but not amphetamine-treated rats, the deleterious effects on spatial memory were associated with changes in MMP-9 level. Because the GluN2B-containing NMDA receptor dominates in adolescence, mephedrone seems to induce more harmful effects on cognition than amphetamine does during this period of life.

Response of immature rats to a low dose of nanoparticulate silver: Alterations in behavior, cerebral vasculature-related transcriptome and permeability.

The increasing production and use of silver nanoparticles (AgNPs) as antimicrobial agents in medicinal and commercial products creates a substantial risk of exposure, especially for infants and children. Our current knowledge concerning the impact of AgNPs on developing brain is insufficient. Therefore we investigated the temporal profile of transcriptional changes in cellular components of the neurovascular unit in immature rats exposed to a low dose of AgNPs. The behavior of animals under these conditions was also monitored. Significant deposition of AgNPs in brain of exposed rats was identified and found to persist over the post-exposure time. Substantial changes were noted in the transcriptional profile of tight junction proteins such as occludin and claudin-5, and pericyte-related molecules such as angiopoietin-1. Moreover, downregulation of platelet-derived growth factor (PDGFß) and its receptor (PDGFßR) which constitute the main signaling pathway between endothelial cells and pericytes was observed. These were long-lasting effects, accompanied by overexpression of astroglial-specific GFAP mRNA and endothelial cell adhesion molecule, ICAM-1, involved in the pathomechanism of neuroinflammation. The profile of changes indicates that even low doses of AgNPs administered during the early stage of life induce dysregulation of neurovascular unit constituents which may lead to disintegration of the blood-brain barrier. This was confirmed by ultrastructural analysis that revealed enhanced permeability of cerebral microvessels resulting in perivascular edema. Changes in the behavior of exposed rats indicating pro-depressive and anti-anxiety impacts were also identified. The results show a high risk of using AgNPs in medical and consumer products dedicated for infants and children.

Identification of a Potent and Selective 5-HT1A Receptor Agonist with In Vitro and In Vivo Antinociceptive Activity.

Opioids are the gold standard drugs for the treatment of acute and chronic severe pain, although their serious side effects constitute a big limitation. In the search for new and safer drugs, 5-HT1AR agonists are emerging as potential candidates in pain relief therapy. In this work, we evaluated the affinity and activity of enantiomers of the two newly synthesized, potent 5-HT1AR agonists N-[(2,2-diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-(pyridin-4-yl)phenoxy]ethan-1-ammonium hydrogenoxalate (rac-1) and N-((2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-methyl-1H-imidazol-5-yl)phenoxy)ethan-1-ammonium hydrogenoxalate (rac-2) in vitro and in vivo. The role of chirality in the interaction with 5-HT1AR was evaluated by molecular docking. The activity of the rac-1 was tested in mouse models of acute pain (hot plate) and severe tonic nociceptive stimulation (intraplantar formalin test). Rac-1 was active in the formalin test with a reduction in paw licking in both phases at 10 mg/kg, and its effect was abolished by the selective 5-HT1AR antagonist, WAY-100635. The eutomer (S)-1, but not the racemate, was active during the hot plate test at 10 and 20 mg/kg, and this effect was abolished by 30 min treatment with WAY-100635 at 30 min. Similarly to 8-OH-DPAT, (S)-1 evoked a slow outward current and depressed spontaneous glutamatergic transmission in superficial dorsal horn neurons, more effectively than rac-1. The eutomer (S)-1 showed promising developability properties, such as high selectivity over 5-HT subtypes, no interaction with the µ receptors, and low hepato- and cardiotoxicity. Therefore, (S)-1 may represent a potential candidate for the treatment of acute and chronic pain without having the adverse effects that are commonly associated with the classic opioid drugs.

Novel Positive Allosteric Modulators of µ Opioid Receptor-Insight from In Silico and In Vivo Studies.

Opioids are the drugs of choice in severe pain management. Unfortunately, their use involves serious, potentially lethal side effects. Therefore, efforts in opioid drug design turn toward safer and more effective mechanisms, including allosteric modulation. In this study, molecular dynamics simulations in silico and 'writhing' tests in vivo were used to characterize potential allosteric mechanism of two previously reported compounds. The results suggest that investigated compounds bind to µ opioid receptor in an allosteric site, augmenting action of morphine at subeffective doses, and exerting antinociceptive effect alone at higher doses. Detailed analysis of in silico calculations suggests that first of the compounds behaves more like allosteric agonist, while the second compound acts mainly as a positive allosteric modulator.

The Antipsychotic D2AAK1 as a Memory Enhancer for Treatment of Mental and Neurodegenerative Diseases.

The treatment of memory impairments associated with the central nervous system diseases remains an unmet medical need with social and economic implications. Here we show, that a multi-target ligand of aminergic G protein-coupled receptors with antipsychotic activity in vivo (D2AAK1) stimulates neuron growth and survival and promotes neuron integrity. We focused on the multilevel evaluation of the D2AAK1-related effects on neurons in terms of behavioral, cellular, molecular, and biochemical features in vivo and in vitro, such as memory-related responses, locomotor activity, tissue sections analysis, metabolic activity, proliferation level, neurons morphology, and proteins level involved in intracellular signaling pathways. In silico studies indicate that activation of calcium/calmodulin-dependent protein kinase I (CaMKI) may underline some of the observed activities of the compound. Furthermore, the compound increases hippocampal neuron proliferation via the activation of neurotrophic factors and cooperating signals responsible for cell growth and proliferation. D2AAK1 improves memory and learning processes in mice after both acute and chronic administration. D2AAK1 also causes an increase in the number of hippocampal pyramidal neurons after chronic administration. Because of its neuroprotective properties and pro-cognitive activity in behavioral studies D2AAK1 has the potential for the treatment of memory disturbances in neurodegenerative and mental diseases.

In Vitro and In Vivo Models for the Investigation of Potential Drugs Against Schizophrenia.

Schizophrenia (SZ) is a complex psychiatric disorder characterized by positive, negative, and cognitive symptoms, and is not satisfactorily treated by current antipsychotics. Progress in understanding the basic pathomechanism of the disease has been hampered by the lack of appropriate models. In order to develop modern drugs against SZ, efficient methods to study them in in vitro and in vivo models of this disease are required. In this review a short presentation of current hypotheses and concepts of SZ is followed by a description of current progress in the field of SZ experimental models. A critical discussion of advantages and limitations of in vitro models and pharmacological, genetic, and neurodevelopmental in vivo models for positive, negative, and cognitive symptoms of the disease is provided. In particular, this review concerns the important issue of how cellular and animal systems can help to meet the challenges of modeling the disease, which fully manifests only in humans, as experimental studies of SZ in humans are limited. Next, it is emphasized that novel clinical candidates should be evaluated in animal models for treatment-resistant SZ. In conclusion, the plurality of available in vitro and in vivo models is a consequence of the complex nature of SZ, and there are extensive possibilities for their integration. Future development of more efficient antipsychotics reflecting the pleiotropy of symptoms in SZ requires the incorporation of various models into one uniting model of the multifactorial disorder and use of this model for the evaluation of new drugs.

G protein-coupled receptor binding and pharmacological evaluation of indole-derived thiourea compounds.

Four 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with the corresponding aryl/alkylisothiocyanates in a medium-polarity solvent. Their structures were confirmed by spectral techniques, and the molecular structure of 3 was determined by X-ray crystal analysis. For all derivatives, the binding affinities at the 5-HT2A and 5-HT2C receptors, as well as their functional activities at the 5-HT1A and D2 receptors, were determined. The arylthioureas 1 and 4 were the most active at the 5-HT1A receptor, showing, at the same time, significant selectivity over the studied 5-HT2 and D2 receptor subtypes. The compounds were tested for their pharmacological activities within the central nervous system in relevant mouse models. The involvement of the serotonergic system in the activity of 1 and 4 was indicated. The antinociceptive action of 4 was linked to its anti-inflammatory activity.

Modification of NO-cGMP Pathway Differentially Affects Diazepam- and Flunitrazepam-Induced Spatial and Recognition Memory Impairments in Rodents.

This study investigated the influence of sildenafil and methylene blue (MB), two modulators of the nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway on amnesic effects of two benzodiazepines (BZs) (diazepam (DZ) and flunitrazepam (FNZ)), in rodents-mice and rats. In the modified elevated plus maze (mEPM) and novel object recognition (NOR) tests, MB given ip at a dose of 5 mg/kg 5 min prior to DZ administration (0.25 or 1 mg/kg, sc) enhanced/induced memory impairment caused by DZ. When MB (2.5, 5, and 10 mg/kg) was applied 5 min prior to FNZ administration (0.05 and 0.1 mg/kg), an effect was opposite and memory impairment induced by FNZ was reduced. When sildenafil (2.5 and 5 mg/kg, ip) was applied 5 min prior to DZ, we observed a reduction of DZ-induced memory deficiency in the mEPM test. A similar effect of sildenafil was shown in the NOR test when the drug was applied at doses of 1.25, 2.5, and 5 mg/kg prior to DZ. In the mEPM test, sildenafil at abovementioned doses had no effects on FNZ-induced memory impairment. In turns, sildenafil administered at doses of 2.5 and 5 mg/kg increased the effect of FNZ on memory impairment in the NOR test. In conclusion, the NO-cGMP pathway is involved differentially into BZs-induced spatial and recognition memory impairments assessed using the NOR and mEPM tests. Modulators of the NO-cGMP pathway affect animal behavior in these tests in a different way depending on what benzodiazepine is applied.