Colony-stimulating factor-1 receptor inhibition attenuates microgliosis and myelin loss but exacerbates neurodegeneration in the chronic cuprizone model.

Multiple sclerosis (MS), especially in its progressive phase, involves early axonal and neuronal damage resulting from a combination of inflammatory mediators, demyelination, and loss of trophic support. During progressive disease stages, a microenvironment is created within the central nervous system (CNS) favoring the arrival and retention of inflammatory cells. Active demyelination and neurodegeneration have also been linked to microglia (MG) and astrocyte (AST)-activation in early lesions. While reactive MG can damage tissue, exacerbate deleterious effects, and contribute to neurodegeneration, it should be noted that activated MG possess neuroprotective functions as well, including debris phagocytosis and growth factor secretion. The progressive form of MS can be modeled by the prolonged administration to cuprizone (CPZ) in adult mice, as CPZ induces highly reproducible demyelination of different brain regions through oligodendrocyte (OLG) apoptosis, accompanied by MG and AST activation and axonal damage. Therefore, our goal was to evaluate the effects of a reduction in microglial activation through orally administered brain-penetrant colony-stimulating factor-1 receptor (CSF-1R) inhibitor BLZ945 (BLZ) on neurodegeneration and its correlation with demyelination, astroglial activation, and behavior in a chronic CPZ-induced demyelination model. Our results show that BLZ treatment successfully reduced the microglial population and myelin loss. However, no correlation was found between myelin preservation and neurodegeneration, as axonal degeneration was more prominent upon BLZ treatment. Concomitantly, BLZ failed to significantly offset CPZ-induced astroglial activation and behavioral alterations. These results should be taken into account when proposing the modulation of microglial activation in the design of therapies relevant for demyelinating diseases. Cover Image for this issue: https://doi.org/10.1111/jnc.15394.

The Role of Galectin-3: From Oligodendroglial Differentiation and Myelination to Demyelination and Remyelination Processes in a Cuprizone-Induced Demyelination Model.

The aim of this work was to combine our previously published results with our new data to show how galectin-3 (Gal-3) controls myelin integrity and function, promotes oligodendroglial cell differentiation, and regulates microglial responses to limit cuprizone- (CPZ)-induced demyelination and foster remyelination. In this study, 8-week-old Gal-3-deficient (Lgals3 -/-) and wild type (WT) mice were fed a diet containing 0.2 % CPZ w/w for 6 weeks, after which CPZ was withdrawn in order to allow remyelination. Our results show that remyelination was less efficient in Lgals3 -/- than in WT mice. Electron microscopic images from remyelinated sections in Lgals3 -/- mice revealed collapsed axons with a defective myelin wrap, while remyelinated WT mice had normal axons without relevant myelin wrap disruption. MMP-3 expression increased during remyelination in WT but not in Lgals3 -/- mice. The number of CD45+, TNFα+ and TREM-2b+ cells decreased only in WT mice only, with no alterations in Lgals3 -/- mice during demyelination and remyelination. Therefore, Gal-3 influences remyelination by mechanisms involving the tuning of microglial cells, modulation of MMP activity, and changes in myelin architecture.

3,3-Dibromoflavanone, a synthetic flavonoid derivative for pain management with antidepressant-like effects and fewer side effects than those of morphine in mice.

In the pursuit of new lead compounds with fewer side effects than opioids, the novel synthetic phytochemical core, 3,3-dibromoflavanone (3,3-DBF), has emerged as a promising candidate for pain management. Acute assays demonstrated dose-dependent central and peripheral antinociceptive activity of 3,3-DBF through the µ-opioid receptor. This study aimed to explore repeated administration effects of 3,3-DBF in mice and compare them with morphine. Mice were treated with 3,3-DBF (30 mg/kg), morphine (6 mg/kg), or vehicle for 10 days, alongside single-treatment groups. Unlike morphine, 3,3-DBF demonstrated antinociceptive effects in the hot plate test without inducing tolerance. Locomotor activity and motor coordination tests (evaluated through the inverted screen and rotarod tests) revealed no significant differences between the 3,3-DBF-treated and control groups. The gastrointestinal transit assay indicated that 3,3-DBF did not induce constipation, in contrast to morphine. Furthermore, withdrawal signs assessed with the Gellert-Holtzman scale were not comparable to morphine. Additionally, 3,3-DBF exhibited antidepressant-like activity, reducing immobility time in the forced swimming and tail suspension tests, akin to imipramine. In summary, 3,3-DBF demonstrated antinociceptive effects without inducing tolerance or dependence and exhibited antidepressant properties. These findings highlight the potential of 3,3-DBF as a promising therapeutic agent for pain management and its comorbidities, offering advantages over morphine by minimizing side effects.

Synthesis and anticonvulsant activity of bioisosteres of trimethadione, N-derivative-1,2,3-oxathiazolidine-4-one-2,2-dioxides from α-hydroxyamides.

The synthesis and anticonvulsant activity of novel heterocycles N-derivative-1,2,3-oxathiazolidine-4-one-2,2-dioxides, bioisosteres of trimethadione (TMD, oxazolidine-2,4-dione) and phenytoin (PHE), are described. TMD is an anticonvulsant drug widely used against absences seizures in the early 80's and PHE is an antiepileptic drug with a wide spectrum activity. The intermediates of synthesis of N-derivative-1,2,3-oxathiazolidine-4-one-2,2-dioxides, α-hydroxyamides, were obtained using microwave assisted synthesis. Anticonvulsant screening was performed in mice after intraperitoneal administration in the maximal electroshock seizure test (MES) and subcutaneous pentylenetetrazole seizures test (scPTZ). These new compounds showed a wide spectrum activity and were no neurotoxic in the RotoRod test. α-Hydroxyamides and N-derivative-1,2,3-oxathiazolidine-4-one-2,2-dioxides were 3-4700 times more potent than valproic acid in the MES test. Quantification of anticonvulsant protection was calculated (ED(50)) for the most active candidates; α-hydroxyamides 3a-c and 3e, and N-derivative-oxathiazolidine-4-one-2,2-dioxides 5a-c with ED(50) values of 9.1, 53.9, 44.6, 25.2, 15.1, 91.1 and 0.06mg/kg, respectively, in the MES test.

Biological Evaluation of Valeriana Extracts from Argentina with Potent Cholinesterase Inhibition for the Treatment of Neurodegenerative Disorders and Their Comorbidities-The Case of Valeriana carnosa Sm. (Caprifoliaceae) Studied in Mice.

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathophysiology includes the abnormal accumulation of proteins (e.g., ß-amyloid), oxidative stress, and alterations in neurotransmitter levels, mainly acetylcholine. Here we present a comparative study of the effect of extracts obtained from endemic Argentinian species of valerians, namely V. carnosa Sm., V. clarionifolia Phil. and V. macrorhiza Poepp. ex DC from Patagonia and V. ferax (Griseb.) Höck and V. effusa Griseb., on different AD-related biological targets. Of these anxiolytic, sedative and sleep-inducing valerians, V. carnosa proved the most promising and was assayed in vivo. All valerians inhibited acetylcholinesterase (IC50 between 1.08-12.69 mg/mL) and butyrylcholinesterase (IC50 between 0.0019-1.46 mg/mL). They also inhibited the aggregation of ß-amyloid peptide, were able to chelate Fe2+ ions, and exhibited a direct relationship between antioxidant capacity and phenolic content. Moreover, V. carnosa was able to inhibit human monoamine oxidase A (IC50: 0.286 mg/mL (0.213-0.384)). A daily intake of aqueous V. carnosa extract by male Swiss mice (50 and 150 mg/kg/day) resulted in anxiolytic and antidepressant-like behavior and improved spatial memory. In addition, decreased AChE activity and oxidative stress markers were observed in treated mouse brains. Our studies contribute to the development of indigenous herbal medicines as therapeutic agents for AD.

2'-Nitroflavone induces apoptosis and modulates mitogen-activated protein kinase pathways in human leukaemia cells.

The cytotoxic activity of 2'-nitroflavone was evaluated in different haematological cancer cell lines and its mechanism of action was further studied in HL-60 cells. 2'-Nitroflavone arrested the cell cycle at the G(2)/M phase and induced an apoptotic response characterized by an increase in the sub-G1 fraction of cells, a typical DNA ladder fragmentation, chromatin condensation and the detection of cells stained with Annexin V. Apoptosis was dependent on the activation of at least caspase-8, caspase-9 and caspase-3. The involvement of the death receptor pathway was indicated by the upregulation of both the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5). We also showed that 2'-nitroflavone increased the expression levels of Bax and induced the release of cytochrome C to cytosol, suggesting the participation of the mitochondria-dependent pathway. When mitogen-activated protein kinases pathways were studied, it was found that p38 and c-Jun NH(2)-terminal kinase (JNK) pathways were activated by 2'-nitroflavone in HL-60 cells, whereas the phosphorylation levels of extracellular signal-regulated kinases (ERK) 1/2 decreased significantly. In addition, whereas both pharmacological inhibition of JNK and downregulation of JNK expression by RNA interference reduced the nitroflavone growth-inhibitory activity and the apoptotic effect, contrasting results were obtained when the ERK1/2 pathway was inhibited, and no effect was observed in the presence of a specific inhibitor of p38 mitogen-activated protein kinase. These findings show for the first time the antitumour action of 2'-nitroflavone in haematological cancer cell lines and suggest that both JNK and ERK1/2 cascades are involved in the apoptotic response induced by 2'-nitroflavone in HL-60 cells.

Chronic intraperitoneal and oral treatments with hesperidin induce central nervous system effects in mice.

Hesperidin (HN) is a flavanone glycoside abundantly found in citrus fruits. This flavonoid mediated central nervous system activity following intraperitoneal (i.p.) acute treatment. The responses of mice after the chronic i.p. (4 and 30 mg/kg/day) or the oral intake administration of this drug (20, 50 and 100 mg/kg/day) were studied by using the holeboard, the plus-maze and the locomotor activity tests. Hesperidin, chronically administered by the i.p. route, exerted a decrease in the locomotor and exploratory activities, thus evidencing a depressant activity. In turn, the chronic oral intake of this flavonoid induced anxiolytic-like effects. These varied responses could be attributed to the different routes of administration that could lead to the production of diverse active metabolites.

Multitarget 2'-hydroxychalcones as potential drugs for the treatment of neurodegenerative disorders and their comorbidities.

The complex nature of neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD) and Parkinson's disease (PD) calls for multidirectional treatment. Restoring neurotransmitter levels by combined inhibition of cholinesterases (ChEs) and monoamine oxidases (MAOs, MAO-A and MAO-B), in conjunction with strategies to counteract amyloid ß (Aß) aggregation, may constitute a therapeutically strong multi-target approach for the treatment of NDDs. Chalcones are a subgroup of flavonoids with a broad spectrum of biological activity. We report here the synthesis of 2'-hydroxychalcones as MAO-A and MAO-B inhibitors. Compounds 5c (IC50 = 0.031 ± 0.001 µM), 5a (IC50 = 0.084 ± 0.003 µM), 2c (IC50 = 0.095 ± 0.019 µM) and 2a (IC50 = 0.111 ± 0.006 µM) were the most potent, selective and reversible inhibitors of human (h)MAO-B isoform. hMAO-B inhibitors 1a, 2a and 5a also inhibited murine MAO-B in vivo in mouse brain homogenates. Molecular modelling rationalised the binding mode of 2'-hydroxychalcones in the active site of hMAO-B. Additionally, several derivatives inhibited murine acetylcholinesterase (mAChE) (IC50 values from 4.37 ± 0.83 µM to 15.17 ± 6.03 µM) and reduced the aggregation propensity of Aß. Moreover, some derivatives bound to the benzodiazepine binding site (BDZ-bs) of the γ-aminobutyric acid A (GABAA) receptors (1a and 2a with Ki = 4.9 ± 1.1 µM and 5.0 ± 1.1 µM, respectively), and exerted sedative and/or anxiolytic like effects on mice. The biological results reported here on 2'-hydroxychalcones provide an extension to previous studies on chalcone scaffold and show them as a potential treatment strategy for NDDs and their associated comorbidities.

Longitudinal evaluation of a novel BChE PET tracer as an early in vivo biomarker in the brain of a mouse model for Alzheimer disease.

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aß) deposition, longitudinally assessed by [18F]florbetaben-PET imaging. Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aß plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aß at 12 months on brain samples. Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aß plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age. Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aß accumulation, suggesting that BChE is a promising early biomarker for incipient AD.

Neurobehavioral evaluation and phytochemical characterization of a series of argentine valerian species.

Folkloric or galenic preparations of valerian roots and rhizomes have been used as sedatives/anxiolytics and sleep inducers since ancient times. "Valerianas" are plants that naturally grow in our region. Although some of them are used in folk medicine, they lack scientific information. We performed a comparative study of the phytochemical composition and the potential in vivo effects of ethanolic extracts of argentine valerian species: Valeriana carnosa Sm., V. clarionifolia Phil. and V. macrorhiza Poepp. ex DC., from "Patagonia Argentina"; V. ferax (Griseb.) Höck and V. effusa Griseb., from the central part of our country, and V. officinalis (as the reference plant). All these plants were rich in phenolic compounds, evidenced the presence of ligands for the benzodiazepine binding site of the GABAA receptor and were able to induce sedation as assessed by loss-of-righting reflex assays (500 mg/kg, i.p.). Mice treated with V. macrorhiza, V. carnosa and V. ferax extracts showed reduced exploratory behaviors while V. clarionifolia produced anxiolytic-like activities (500 mg/kg, i.p.) in the Hole board test. Oral administrations (300 mg/kg and 600 mg/kg, p.o.) evidenced sedative effects for V. ferax and anxiolytic-like properties for V. macrorhiza, V. carnosa and V. clarionifolia extracts. Our native valerian species are active on the CNS, validating its folkloric use as anxiolytic/sedative and sleep enhancers.

Stereoselective Activity of 1-Propargyl-4-styrylpiperidine-like Analogues That Can Discriminate between Monoamine Oxidase Isoforms A and B.

The resurgence of interest in monoamine oxidases (MAOs) has been fueled by recent correlations of this enzymatic activity with cardiovascular, neurological, and oncological disorders. This has promoted increased research into selective MAO-A and MAO-B inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be achieved by geometric isomers of cis- and trans-1-propargyl-4-styrylpiperidines. While the cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only the MAO-B isoform. The inhibition was studied by kinetic analysis, UV-vis spectrum measurements, and X-ray crystallography. The selective inhibition of the MAO-A and MAO-B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous system disorders. This study represents a unique case of stereoselective activity of cis/trans isomers that can discriminate between structurally related enzyme isoforms.

In vitro induction of apoptosis and in vivo effects of a flavone nitroderivative in murine mammary adenocarcinoma cells.

We explored the in vitro and in vivo mechanism of antitumor action of the synthetic flavonoid 2'-nitroflavone on LM3 murine mammary adenocarcinoma cells. In vitro assays showed that 2'-nitroflavone increased the population of LM3 hypodiploid cells and produced a typical ladder of DNA fragmentation. Apoptotic cell death was also characterized by the activation of caspase-8, -9 and -3, by an increment in the expression levels of the proapoptotic protein Bax and by the release of cytochrome c to cytosol. The in vivo effect of 2'-nitroflavone on tumor growth was studied in BALB/c mice injected subcutaneously with LM3 cells. Results showed that tumor volume and weight were significantly reduced at doses of 10 and 40 mg/kg of 2'-nitroflavone, respectively. Apoptotic cells were identified by TUNEL assay in tumor slices from mice treated with 10 mg/kg of 2'-nitroflavone. Western blot analysis of tumor lysate supernatants from treated mice revealed an upregulation of the total levels of Bax and Fas receptor. In addition, administration of 40 mg/kg of 2'-nitroflavone to nontumor-bearing mice showed no histopathological effects on different organ tissues. This is the first report of the in vivo growth inhibitory effect of 2'-nitroflavone as an apoptotic agent likely useful for mammary adenocarcinoma treatment.

Hesperidin, a flavonoid glycoside with sedative effect, decreases brain pERK1/2 levels in mice.

The aim of this work was to evaluate if the intraperitoneal administration of the natural compound hesperidin, in a sedative dose, and neo-hesperidin, a hesperidin structural analog that exerts minor sedative effect, were able to induce changes in intracellular signaling cascades in different areas of the brain. The systemic administration of hesperidin produced a marked reduction in the phosphorylation state of extracellular signal-regulated kinases 1/2 (ERK 1/2), but not of Ca(+2)/calmodulin-dependent protein kinase II alpha subunit (alphaCaMKII), in the cerebral cortex, cerebellum and hippocampus. In contrast, neo-hesperidin did not markedly affect the activity of ERK 1/2 in both the cortex and the cerebellum. Taken together, these results demonstrated that intracellular signalling involving a selective decrease in ERK1/2 activation accompanied the depressant action of hesperidin. Even more, the low sedative action of neo-hesperidin correlates with a negligible decrease in phosphorylation state of ERK 1/2 (pERK 1/2), suggesting that low levels of pERK 1/2 in CNS could be a marker of sedative efficacy of flavonoids.

Neuroactive flavonoid glycosides from Tilia petiolaris DC. extracts.

Pharmacological assay guided purification of an ethanol extract of Tilia petiolaris DC. inflorescences resulted in the isolation and identification of isoquercitrin (ISO), quercetin 3-O-glucoside-7-O-rhamnoside (QUE) and kaempferol 3-O-glucoside-7-O-rhamnoside (KAE). The behavioral actions of these glycosylated flavonoids were examined in the hole board, locomotor activity and thiopental-induced loss of righting reflex tests in mice. QUE (10 and 30 mg/kg) and KAE (30 mg/kg), intraperitoneally (i.p.) administered to mice, reduced all the parameters measured in the hole board test, but ISO (30 mg/kg) only reduced the number of rearings. Meanwhile QUE at 30 mg/kg i.p. also decreased the ambulatory locomotor activity and increased the sodium thiopental-induced time of loss of the righting reflex suggesting a clear depressant action. The above results demonstrate the occurrence of neuroactive flavonoid glycosides in Tilia.

Chalcone derivatives: synthesis, in vitro and in vivo evaluation of their anti-anxiety, anti-depression and analgesic effects.

Anxiety disorders, depression and pain are highly prevalent pathologies. Their pharmacotherapy is associated with unwanted side effects; hence there is a clinical need to develop more effective drugs with fewer adverse reactions. Chalcones are one of the major classes of naturally occurring compounds. Chalcones and their derivatives have a huge importance in medicinal chemistry, displaying a wide range of pharmacological activities including anti-inflammatory, antimicrobial, antioxidant, cytotoxic and antitumor actions. The aim of this work was to evaluate chalcone effects on different targets involved in these pathologies. We have synthesized a series of simple chalcone derivatives taking common structural requirements described in literature related to their anxiolytic-like, antidepressant-like and/or antinociceptive properties into account. Furthermore, their potential in vitro effects towards different targets involved in these pathologies were evaluated. We have obtained twenty chalcones with moderate to high yields and assessed their ability to bind distinctive receptors, from rat brain homogenates, by displacement of labelled specific ligands: [3H] FNZ (binding site of benzodiazepines/GABAA), [3H] 8-OH-DPAT (serotonin 5-HT1A) and [3H] DAMGO (µ-opioid). Those compounds that showed the better in vitro activities were evaluated in mice using different behavioural tasks. In vivo results showed that 5'-methyl-2'-hydroxychalcone (9) exerted anxiolytic-like effects in mice in the plus maze test. While chalcone nuclei (1) revealed antidepressant-like activities in the tail suspension test. In addition, the novel 5'-methyl-2'-hydroxy-3'-nitrochalcone (12) exhibited antinociceptive activity in acute chemical and thermal nociception tests (writhing and hot plate tests). In conclusion, chalcones are thus promising compounds for the development of novel drugs with central nervous system (CNS) actions.

2'-Nitroflavone induces cell cycle arrest and apoptosis in HeLa human cervical carcinoma cells.

The mechanism of antitumor action of a synthetic nitroflavone derivative, 2'-nitroflavone, was evaluated in vitro in HeLa human cervix adenocarcinoma cells. We showed that the nitroflavone derivative slowed down the cell cycle at the S phase and increase the population of cells at the G2/M phase after 24h of incubation. The treatment with 2'-nitroflavone also induced an apoptotic response, characterized by an increase of the sub-G1 fraction of cells, by cells with chromatin condensation and membrane blebbing, by a typical ladder of DNA fragmentation and by detection of apoptotic cells stained with Annexin V. The observed apoptosis was regulated by caspase-8 and -9, both contributing to the activation of the effector caspase-3. In addition, inhibitors of caspase-8 or -9 partially protected HeLa cells from 2'-nitroflavone-induced cell death. We also found that 2'-nitroflavone did not affect the total amount of Bax and Bcl-2 proteins, although a translocation of Bax from cytosol to mitochondria was evident after 6h of exposure. Furthermore, 2'-nitroflavone decreased the expression of the anti-apoptotic Bcl-XL protein, induced the release of cytochrome C to cytosol and increased the levels of Fas and Fas-L. Our results indicated that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2'-nitroflavone and suggest that this derivative could be a potentially useful agent for the treatment of certain malignancies.

Anticonvulsant and anxiolytic-like effects of compounds isolated from Polygala sabulosa (Polygalaceae) in rodents: in vitro and in vivo interactions with benzodiazepine binding sites.

RATIONALE: Polygala sabulosa, a folk medicine, presents dihydrostyryl-2-pyrones (DST) and styryl-2-pyrones (STY), compounds structurally similar to kavalactones. Our previous study showed that the ethyl acetate fraction (EA) and these constituents present anxiolytic-like, hypno-sedative, and anticonvulsant effects in mice. OBJECTIVES: This study investigated the role of benzodiazepine binding site (BDZ-bs) in the central effects of either EA or three DST (1, 2, and 3) and three STY (4, 5, and 7), using in vivo and in vitro assays. METHODS AND RESULTS: In the elevated plus-maze (EPM), flumazenil (FMZ), a BDZ antagonist, partially blocked the anxiolytic-like effect of DST-3 or STY-4 and STY-7, but not DST-1. Using electroencephalogram (EEG), EA protected against pentylenetetrazole (PTZ)-induced convulsion in rats, an effect partially blocked by FMZ, suggesting the participation of the BDZ-bs in this action. EA also protected against the maximal electroshock (MES)-induced convulsions in mice, a profile distinct from diazepam (DZP). DST and STY compounds inhibited the [(3)H]-flunitrazepam ([(3)H]-FNZ) binding to BDZ-bs in rat cortical synaptosomes with K (i) higher than 100 microM (DST-1), 41.7 microM (DST-2), 35.8 microM (DST-3), 90.3 microM (STY-4), 31.0 microM (STY-5) and 70.0 microM (STY-7). In the saturation assay, DST-3 and STY-7 competitively inhibited the binding of [(3)H]-FNZ to BDZ-bs with a significant decrease in apparent affinity (K (d)) and no change in maximal binding (B (max)). CONCLUSIONS: The present data support a partial BDZ-bs mediation of the anxiolytic-like and anticonvulsant effects of EA of P. sabulosa and its main isolated constituents, DST and STY.

Opioid receptors are involved in the sedative and antinociceptive effects of hesperidin as well as in its potentiation with benzodiazepines.

Previous reports from our laboratory described the sedative activity of hesperidin (hesperetin-7-rhamnoglucoside). This property is greatly increased when the glycoside is injected jointly with diazepam and this interaction has been shown to be synergistic. In the present work the generality of the synergistic phenomenon is proved, since potentiation also occurs with several other benzodiazepines, namely alprazolam, bromazepam, midazolam and flunitrazepam. In order to advance in the study of the mechanism of action of hesperidin, the possible participation of several brain receptors, which are implicated in the control of numerous behavioral and physiological functions, was explored by investigating the effects of a variety of their antagonists on hesperidin actions. The results showed that the 5-HT2 receptor and the alpha1-adrenoceptor seem unlikely to be involved in the behavioral effects of hesperidin. Naltrexone, a nonselective antagonist of opioid receptors, totally blocked hesperidin effects on locomotion, and partially antagonized hesperidin-induced decreased exploration in the hole board test. Nor-binaltorphimine, a selective kappa opioid receptor antagonist, was able to partially block hesperidin effects on locomotor activity. Furthermore, hesperidin-induced antinociception was partially blocked by naltrexone, and potentiated by co-administration with alprazolam. Hence, the participation of the opioid system in the sedative, antinociceptive and potentianting effects of hesperidin with benzodiazepines in mice is highly probable. Our results suggest a possible beneficial use of the association of hesperidin with benzodiazepines, not only to improve human sedative therapy, but also in the management of pain.

6,3'-Dinitroflavone is a low efficacy modulator of GABA(A) receptors.

6,3'-Dinitroflavone (6,3'-DNF) is a synthetic flavone derivative that exerts anxiolytic effects in the elevated plus maze. Based on the finding that this effect is blocked by Ro15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate) which is a specific antagonist at the benzodiazepine binding site of GABA(A) receptors we investigated the interaction of 6,3'-DNF with several recombinant GABA(A) receptor subtypes. Inhibition of [(3)H]flunitrazepam binding to recombinant GABA(A) receptors in transiently transfected HEK293 cells indicated that 6,3'-DNF exhibited the highest affinity for GABA(A) receptors composed of alpha1beta2gamma2 subunits and a 2-20 fold lower affinity for homologous receptors containing alpha2, alpha3, or alpha5 subunits. Two-electrode voltage-clamp experiments in Xenopus oocytes indicated that 6,3'-DNF does not induce chloride flux in the absence of GABA, but exerts low efficacy inverse agonistic modulatory effects on GABA-elicited currents in the GABA(A) receptor subtypes alpha1beta2gamma2 and alpha5beta2gamma2. In the subtypes alpha2beta2gamma2, alpha3beta2gamma2, alpha4beta2gamma2, alpha6beta2gamma2 or alpha4beta2delta and alpha4beta3delta, 6,3'-DNF exerts either none or very low efficacy positive modulatory effects. In contrast, 100 nM Ro15-1788 exhibited weak to moderate partial agonistic effects on each receptor investigated. These data indicate that Ro15-1788 only can antagonize the weak inverse agonist effects of 6,3'-DNF on alpha1beta2gamma2 and alpha5beta2gamma2 receptors, but will enhance the weak agonistic effects on the other receptor subtypes investigated. The possible mechanism of the Ro15-1788 sensitive anxiolytic effect of 6,3'-DNF is discussed.

N-propyl-2,2-diphenyl-2-hydroxyacetamide, a novel α-hydroxyamide with anticonvulsant, anxiolytic and antidepressant-like effects that inhibits voltage-gated sodium channels.

In patients with epilepsy, anxiety and depression are the most frequent psychiatric comorbidities but they often remain unrecognized and untreated. We report herein the antidepressant-like activity in two animal models, tail suspension and forced swimming tests, of six anticonvulsants α-hydroxyamides. From these, N-propyl-2,2-diphenyl-2-hydroxyacetamide (compound 5) emerged not only as the most active as anticonvulsant (ED50 = 2.5mg/kg, MES test), but it showed the most remarkable antidepressant-like effect in the tail suspension and forced swimming tests (0.3-30mg/kg, i.p.); and, also, anxiolytic-like action in the plus maze test (3-10mg/kg, i.p.) in mice. Studies of its mechanism of action, by means of its capacity to act via the GABAA receptor ([3H]-flunitrazepam binding assay); the 5-HT1A receptor ([3H]-8-OH-DPAT binding assay) and the voltage-gated sodium channels (either using the patch clamp technique in hNav 1.2 expressed in HEK293 cell line or using veratrine, in vivo) were attempted. The results demonstrated that its effects are not likely related to 5-HT1A or GABAAergic receptors and that its anticonvulsant and antidepressant-like effect could be due to its voltage-gated sodium channel blocking properties.