The effect of diazepam (1.0 mg/kg, i.p.) and buspirone (5.0 mg/kg, i.p.) on the burying behaviour latency (denoting actions on the animals' reactivity) and on the cumulative burying behaviour (directly reflecting the experimental anxiety levels), were analyzed in male-, intact females, at proestrus and metoestrus, and in neonatally-androgenized-rats. Androgenization was performed by injecting 60 micrograms/rat of testosterone propionate on day 5 after delivery. Two main groups of neonatally-androgenized rats were established: A group of animals showing permanent oestrus from the vaginal opening (acyclic females) and a group presenting the delayed anovulatory syndrome. Diazepam produced a clear reduction in experimental anxiety in males and neonatally-androgenized-females. Particularly important was the anxiolytic effect of diazepam on acyclic females that was accompanied by a significant increase in burying behaviour latency. Conversely, buspirone induced a clear reduction in burying behaviour, without modifying its latency, in all groups regardless of the gender and the neonatal treatment. Data are discussed on the basis of the androgen participation on the anxiolytic drug effects. A possible age-related benzodiazepine actions in females is suggested.

Pharmacological stimulation/antagonism of astrocyte glio-peptide octadecaneuropeptide signaling alters ventromedial hypothalamic nucleus (VMN) counterregulatory γ-aminobutyric acid (GABA) and nitric oxide transmission. The current research used newly developed capillary zone electrophoresis-mass spectrometry methods to investigate hypoglycemia effects on VMN octadecaneuropeptide content, along with gene knockdown tools to determine if octadecaneuropeptide signaling regulates these transmitters during eu- and/or hypoglycemia. Hypoglycemia caused dissimilar adjustments in the octadecaneuropeptide precursor, i.e., diazepam-binding-inhibitor and octadecaneuropeptide levels in dorsomedial versus ventrolateral VMN. Intra-VMN diazepam-binding-inhibitor siRNA administration decreased baseline 67 and 65 kDa glutamate decarboxylase mRNA levels in GABAergic neurons laser-microdissected from each location, but only affected hypoglycemic transcript expression in ventrolateral VMN. This knockdown therapy imposed dissimilar effects on eu- and hypoglycemic glucokinase and 5'-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) gene profiles in dorsomedial versus ventrolateral GABAergic neurons. Diazepam-binding-inhibitor gene silencing up-regulated baseline (dorsomedial) or hypoglycemic (ventrolateral) nitrergic neuron neuronal nitric oxide synthase mRNA profiles. Baseline nitrergic cell glucokinase mRNA was up- (ventrolateral) or down- (dorsomedial) regulated by diazepam-binding-inhibitor siRNA, but knockdown enhanced hypoglycemic profiles in both sites. Nitrergic nerve cell AMPKα1 and -α2 transcripts exhibited division-specific responses to this genetic manipulation during eu- and hypoglycemia. Results document the utility of capillary zone electrophoresis-mass spectrometric tools for quantification of ODN in small-volume brain tissue samples. Data show that hypoglycemia has dissimilar effects on ODN signaling in the two major neuroanatomical divisions of the VMN and that this glio-peptide imposes differential control of glucose-regulatory neurotransmission in the VMNdm versus VMNvl during eu- and hypoglycemia.

Acyl-CoA-binding protein (ACBP)/diazepam-binding inhibitor has lately been described as an endocrine factor affecting food intake and lipid metabolism. ACBP is dysregulated in catabolic/malnutrition states like sepsis or systemic inflammation. However, regulation of ACBP has not been investigated in conditions with impaired kidney function, so far.

Benzodiazepine receptor agonists are widely prescribed therapeutic agents, alter gamma-aminobutyric acid (GABA)A receptor function, and have hypnotic, anxiolytic, anticonvulsant, and antispastic effects. GABAA receptor activity increases under systemic inflammatory conditions. We investigated the effect of benzodiazepine receptor agonists on pentobarbital-induced loss of righting reflex (LORR) duration using a mouse model of lipopolysaccharide (LPS)-induced inflammation. We assessed pentobarbital-induced LORR duration 24 h after LPS treatment in mice. Additionally, we examined the microglial response by immunohistochemistry and serum IL-6 and TNF-α concentrations in mice. LPS treatment significantly increased the duration of pentobarbital-induced LORR in mice treated with benzodiazepine receptor agonists (diazepam and brotizolam) and a GABAA receptor agonist (muscimol) compared to that of mice treated with vehicle. These effects were blocked by bicuculline, a GABAA receptor antagonist. LPS significantly increased the number of ionized calcium binding adapter molecule-1-positive hippocampal cells 2 and 24 h after treatment. The enhancing effect of diazepam in LPS-treated mice was significantly reduced by minocycline. These findings suggest that LPS enhances pentobarbital-induced LORR duration in mice treated with benzodiazepine via GABAA receptor activity.

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

Benzodiazepines (BZDs) are a class of widely prescribed psychotropic drugs that modulate activity of GABAA receptors (GABAARs), neurotransmitter-gated ion channels critical for synaptic transmission. However, the physical basis of this modulation is poorly understood. We explore the role of an important gating domain, the α1M2-M3 linker, in linkage between the BZD site and pore gate. To probe energetics of this coupling without complication from bound agonist, we use a gain of function mutant (α1L9'Tβ2γ2L) directly activated by BZDs. We identify a specific residue whose mutation (α1V279A) more than doubles the energetic contribution of the BZD positive modulator diazepam (DZ) to pore opening and also enhances DZ potentiation of GABA-evoked currents in a wild-type background. In contrast, other linker mutations have little effect on DZ efficiency, but generally impair unliganded pore opening. Our observations reveal an important residue regulating BZD-pore linkage, thereby shedding new light on the molecular mechanism of these drugs.

Benzodiazepines facilitate the inhibitory actions of GABA by binding to γ-aminobutyric acid type A receptors (GABAARs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time- and dose-dependent decrease in surface levels of GABAARs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABAAR activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABAARs, involving mobilisation of Ca2+ from the intracellular stores and activation of the Ca2+/calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABAARs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABAARs and Ca2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCδ, a PLC isoform found in direct association with GABAARs. Thus, a PLCδ/Ca2+/calcineurin signalling cascade converts the initial enhancement of GABAARs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.

The goal of the present study was to examine the effects of N-methyl-aspartate (NMDA) receptor antagonists-memantine and ketamine and the drugs modifying the NO:cGMP pathway-NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), the endogenous precursor of NO-L-arginine, and the guanylyl cyclase inhibitor-methylene blue (MB) on the development of sensitization to withdrawal signs precipitated after chronic, interrupted treatment with diazepam, a benzodiazepine receptor agonist, in mice. To develop the sensitization, the mice were divided into groups: continuously and sporadically (with two diazepam-free periods) treated with diazepam (15 mg/kg, sc). To precipitate the withdrawal syndrome (clonic and tonic seizures, and death), pentylenetetrazole (55 mg/kg, sc) with the benzodiazepine receptor antagonist, flumazenil (5.0 mg/kg, ip), were administered after the last injection of diazepam or saline. Memantine (2.5, 5.0 mg/kg), and ketamine (2.5, 5.0 mg/kg), L-NAME (100, 200 mg/kg) and 7-NI (20 and 40 mg/kg), L-arginine (250, 500 mg/kg) and MB (5 and 10 mg/kg) were administered ip in sporadically diazepam-treated mice during the diazepam-free periods. Our results indicated that both NMDA receptor antagonists and drugs that inhibit the NO:cGMP pathway, except L-arginine (the endogenous donor of NO), attenuated the diazepam-induced sensitization to withdrawal signs in mice. Thus, NMDA receptors and the NO:cGMP pathway are involved in the mechanisms of sensitization to benzodiazepine withdrawal.

Constant rate infusion (CRI) of benzodiazepines or propofol (PPF) is a therapeutic option for cluster seizures (CS) and status epilepticus (SE) in canine patients non-responding to first-line benzodiazepines or non-anesthetics. However, specific indications for optimal duration of CRI are lacking. The aim of this study was to determine the effect of duration of anesthetic CRI on outcome and length of hospital stay in dogs with refractory seizure activity of different etiology.

Previous studies have demonstrated a profound lack of habituation in 129P3 mice compared to the habituating, but initially more anxious, BALB/c mice. The present study investigated whether this non-adaptive phenotype of 129P3 mice is primarily based on anxiety-related characteristics.

Acyl coenzyme A binding protein (ACBP), also known as diazepam binding inhibitor (DBI) is a multifunctional protein with an intracellular action (as ACBP), as well as with an extracellular role (as DBI). The plasma levels of soluble ACBP/DBI are elevated in human obesity and reduced in anorexia nervosa. Accumulating evidence indicates that genetic or antibody-mediated neutralization of ACBP/DBI has anorexigenic effects, thus inhibiting food intake and inducing lipo-catabolic reactions in mice. A number of anorexiants have been withdrawn from clinical development because of their side effects including an increase in depression and suicide. For this reason, we investigated the psychiatric impact of ACBP/DBI in mouse models and patient cohorts. Intravenously (i.v.) injected ACBP/DBI protein conserved its orexigenic function when the protein was mutated to abolish acyl coenzyme A binding, but lost its appetite-stimulatory effect in mice bearing a mutation in the γ2 subunit of the γ-aminobutyric acid (GABA) A receptor (GABAAR). ACBP/DBI neutralization by intraperitoneal (i.p.) injection of a specific mAb blunted excessive food intake in starved and leptin-deficient mice, but not in ghrelin-treated animals. Neither i.v. nor i.p. injected anti-ACBP/DBI antibody affected the behavior of mice in the dark-light box and open-field test. In contrast, ACBP/DBI increased immobility in the forced swim test, while anti-ACBP/DBI antibody counteracted this sign of depression. In patients diagnosed with therapy-resistant bipolar disorder or schizophrenia, ACBP/DBI similarly correlated with body mass index (BMI), not with the psychiatric diagnosis. Patients with high levels of ACBP/DBI were at risk of dyslipidemia and this effect was independent from BMI, as indicated by multivariate analysis. In summary, it appears that ACBP/DBI neutralization has no negative impact on mood and that human depression is not associated with alterations in ACBP/DBI concentrations.

Diazepam has been detected in water sources around the world affecting the quality of drinking water. Even in small quantities, recent studies have proven the negative effects of the drug on human body. Since traditional water and sewage treatment do not remove this type of contaminant, it became interesting to evaluate forms to remove them from water sources. A cheap and eco-friendly alternative to remove this drug from the water is through adsorption using the natural clinoptilolite zeolite as an adsorbent. This work goal was to study the characterizations of clinoptilolite, such as scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffractometer (XRD), and Fourier transform infrared spectroscopy (FTIR) and analyze the potential of this material as an adsorbent. Kinetic studies and isotherm analysis were performed in batch. The results showed the potential of the natural zeolite to remove the pollutant in an aqueous medium reaching a maximum adsorption capacity of 8.25 mg g-1. The adsorption process followed a pseudo-second-order kinetics indicating that the adsorption was based on a chemisorption process. The isotherms curves shown favorable adsorption and the Langmuir isotherm model fit the experimental data better.

Background: Alcohol use disorder (AUD) is characterized by chronic excessive alcohol consumption, often alternating with periods of abstinence known as alcohol withdrawal syndrome (AWS). Diazepam is the preferred benzodiazepine for treatment of alcohol withdrawal syndrome under most circumstances, but the specific mechanism underlying the treatment needs further research. Methods: We constructed an animal model of two-bottle choices and chronic intermittent ethanol exposure. LC-MS/MS proteomic analysis based on the label-free and intensity-based quantification approach was used to detect the protein profile of the whole brain. Weighted gene correlated network analysis was applied for scale-free network topology analysis. We established a protein-protein interaction network based on the Search Tool for the Retrieval of Interacting Genes (STRING) database and Cytoscape software and identified hub proteins by CytoHubba and MCODE plugins of Cytoscape. The online tool Targetscan identified miRNA-mRNA pair interactions. Results: Seven hub proteins (Dlg3, Dlg4, Shank3, Grin2b, Camk2b, Camk2a and Syngap1) were implicated in alcohol withdrawal syndrome or diazepam treatment. In enrichment analysis, glutamatergic synapses were considered the most important pathway related to alcohol use disorder. Decreased glutamatergic synapses were observed in the late stage of withdrawal, as a protective mechanism that attenuated withdrawal-induced excitotoxicity. Diazepam treatment during withdrawal increased glutamatergic synapses, alleviating withdrawal-induced synapse inhibition. Conclusion: Glutamatergic synapses are considered the most important pathway related to alcohol use disorder that may be a potential molecular target for new interventional strategies.

Loss of parvalbumin interneurons in the hippocampus is a robust finding in schizophrenia brains. Rats exposed during embryonic day 17 to methylazoxymethanol acetate exhibit characteristics consistent with an animal model of schizophrenia, including decreased parvalbumin interneurons in the ventral hippocampus. We reported previously that peripubertal administration of diazepam prevented the emergence of pathophysiology in adult methylazoxymethanol acetate rats.

Of the neurotransmitters that influence neurogenesis, gamma-aminobutyric acid (GABA) plays an outstanding role, and GABA receptors support non-synaptic signaling in progenitors and migrating neurons. Here, we report that expression levels of diazepam binding inhibitor (DBI), an endozepine that modulates GABA signaling, regulate embryonic neurogenesis, affecting the long-term outcome regarding the number of neurons in the postnatal mouse brain. We demonstrate that DBI is highly expressed in radial glia and intermediate progenitor cells in the germinal zones of the embryonic mouse brain that give rise to excitatory and inhibitory cells. The mechanism by which DBI controls neurogenesis involves its action as a negative allosteric modulator of GABA-induced currents on progenitor cells that express GABAA receptors containing γ2 subunits. DBI's modulatory effect parallels that of GABAA-receptor-mediating signaling in these cells in the proliferative areas, reflecting the tight control that DBI exerts on embryonic neurogenesis.

No abstract available

NRL-1 is a novel intranasal formulation of diazepam that is being evaluated as rescue medication in patients with epilepsy who experience bouts of increased seizure activity despite stable regimens of antiepileptic drugs. This phase 1, open-label, randomized, crossover study in healthy adult volunteers consisted of 3 single-dose periods (5, 10, and 20 mg) followed by a 2-dose period (2 × 10 mg) with a minimum 28-day washout between treatments. Blood samples were taken at prespecified time points after intranasal dosing, and bioanalytic analysis of diazepam and nordiazepam was conducted using a validated liquid chromatography-tandem mass spectrometry method. Plasma pharmacokinetic parameters were summarized using descriptive statistics, and dose proportionality (peak concentration [Cmax ] and area under the plasma concentration-time curve [AUC0-∞ ]) was evaluated based on a power model within a 90%CI of 0.84 to 1.16. Comparisons were also conducted between single 10-mg dose and multidose (2 × 10 mg) treatments. NRL-1 administration resulted in rapid diazepam absorption (median time to peak concentration 1.4-1.5 hours). Plasma concentration-time profiles showed similar patterns of exposure that appeared to be dose dependent, with Cmax of 85.6, 133.6, and 235.3 ng/mL for the 5-, 10-, and 20-mg doses, respectively, although the lower 90%CI for Cmax and AUC0-∞ exceeded dose proportionality criteria. The coefficient of variation ranged from 59% to 67% for Cmax and 48% to 56% for AUC parameters. Dose-normalized AUC0-∞ values were comparable between the 2 × 10-mg and single 10-mg doses. Treatment-emergent adverse events were consistent with those expected for diazepam, with transient somnolence the most frequent adverse event (94.4%). These results support NRL-1 as a potential therapy for managing seizure emergencies.

The activity of the Schaffer collaterals of hippocampal CA3 neurons and hippocampal CA1 neurons has been shown to increase after fluid percussion injury. Diazepam can inhibit the hyperexcitability of rat hippocampal neurons after injury, but the mechanism by which it affects excitatory synaptic transmission remains poorly understood. Our results showed that diazepam treatment significantly increased the slope of input-output curves in rat neurons after fluid percussion injury. Diazepam significantly decreased the numbers of spikes evoked by super stimuli in the presence of 15 μmol/L bicuculline, indicating the existence of inhibitory pathways in the injured rat hippocampus. Diazepam effectively increased the paired-pulse facilitation ratio in the hippocampal CA1 region following fluid percussion injury, reduced miniature excitatory postsynaptic potentials, decreased action-potential-dependent glutamine release, and reversed spontaneous glutamine release. These data suggest that diazepam could decrease the fluid percussion injury-induced enhancement of excitatory synaptic transmission in the rat hippocampal CA1 area.

Benzodiazepines are from the most common drugs which are used for treatment of anxiety disorders. There are other drugs with antianxiety properties including antihistamines such as hydroxyzine, too. Body of evidence show that co-administration of two drugs which act through different mechanisms, makes the dose of each drug to be reduced, while preserving the desired effect with less adverse drug reactions. The aim of this study was to see whether co-administration of subeffective antianxiety doses of diazepam and hydroxyzine has any antianxiety effect in elevated zero-maze (EZM) in mice.

GABAA receptors are pentameric chloride ion channels that are opened by GABA. We have screened a cell line derived from human glioblastoma, U3047MG, for expression of GABAA receptor subunit isoforms and formation of functional ion channels. We identified GABAA receptors subunit α2, α3, α5, β1, β2, β3, δ, γ3, π, and θ mRNAs in the U3047MG cell line. Whole-cell GABA-activated currents were recorded and the half-maximal concentration (EC₅₀) for the GABA-activated current was 36 μM. The currents were activated by THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and enhanced by the benzodiazepine diazepam (1 μM) and the general anesthetics etomidate and propofol (50 μM). In line with the expressed GABAA receptors containing at least the α3β3θ subunits, the receptors were highly sensitive to etomidate (EC₅₀=55 nM). Immunocytochemistry identified expression of the α3 and β3 subunit proteins. Our results show that the GABAA receptors in the glial cell line are functional and are modulated by classical GABAA receptor drugs. We propose that the U3047MG cell line may be used as a model system to study GABAA receptors function and pharmacology in glial cells.