Neurotoxic effects induced by flunitrazepam and its metabolites in zebrafish: Oxidative stress, apoptosis, and histone hypoacetylation.

Benzodiazepines (BZDs) have been widely detected in aquatic environments, but their neurotoxic effects and potential mechanisms are still unclear. This study focuses on flunitrazepam (FLZ) and its metabolite, 7-aminoflunitrazepam (7-FLZ), as representative psychotropic BZD. We investigated their neurotoxic effects on adult zebrafish following a 30-day exposure to environmentally relevant concentrations. The findings reveal that exposure to these drugs induces anxiety-like and aggressive behaviors in zebrafish. Additionally, notable morphological damage to brain tissue and mitochondrial structures was observed. Through TUNEL staining, an increase in apoptotic cells was detected in the brain tissue of the exposed group, accompanied by marked elevations in ROS and caspase-3/9 levels. The upregulation of apoptosis-related genes Bax, p53, and Bcl-2 confirmed the occurrence of apoptosis. Furthermore, exposure to the drugs resulted in decreased acetylation levels of brain histones H3 and H4. The upregulation of histone deacetylation enzyme genes (HDAC1, HDAC3, HDAC4, and HDAC6) supported this result. Molecular docking results suggest that compared to 7-FLZ, FLZ has a higher binding affinity with HDAC3 and HDAC4, explaining why it causes lower histone acetylation levels. This study in zebrafish elucidates the neurotoxicity and molecular mechanisms induced by FLZ and 7-FLZ, which is significant for further understanding the impact of BZDs on human health and assessing their ecological risks.

Flunitrazepam and its metabolites induced brain toxicity: Insights from molecular dynamics simulation and transcriptomic analysis.

Psychoactive drugs frequently contaminate aquatic environments after human consumption, raising concerns about their residues and ecological harm. This study investigates the effects of flunitrazepam (FLZ) and its metabolite 7-aminoflunitrazepam (7-FLZ), benzodiazepine-class psychoactive drugs, on brain accumulation, blood-brain barrier (BBB), and neuroinflammation of the model organism zebrafish. Molecular dynamics simulation and transcriptome sequencing were used to uncover their toxic mechanisms. Results demonstrate that both FLZ and 7-FLZ can accumulate in the brain, increasing Evans blue levels by 3.4 and 0.8 times, respectively. This increase results from abnormal expression of tight junction proteins, particularly ZO-1 and Occludin, leading to elevated BBB permeability. Furthermore, FLZ and 7-FLZ can also induce neuroinflammation, upregulating TNFα by 91% and 39%, respectively, leading to pathological changes and disrupted intracellular ion balance. Molecular dynamics simulation reveals conformational changes in ZO-1 and Occludin proteins, with FLZ exhibiting stronger binding forces and greater toxicity. Weighted gene co-expression network analysis identifies four modules correlated with BBB permeability and neuroinflammation. KEGG enrichment analysis of genes within these modules reveals pathways like protein processing in the endoplasmic reticulum, NOD-like receptor signaling pathway, and arginine and proline metabolism. This study enhances understanding of FLZ and 7-FLZ neurotoxicity and assesses environmental risks of psychoactive substances. ENVIRONMENTAL IMPLICATION: With the increasing prevalence of mental disorders and the discharge of psychoactive drugs into water, even low drug concentrations (ng/L-µg/L) can pose neurological risks. This study, utilizing molecular dynamic (MD) simulations and transcriptome sequencing, investigate the neurotoxicity and mechanisms of flunitrazepam and 7-aminoflunitrazepam. It reveals that they disrupt the blood-brain barrier in zebrafish and induce neuroinflammation primarily by inducing conformational changes in tight junction proteins. MD simulations are valuable for understanding pollutant-protein interactions. This research offers invaluable insights for the environmental risk assessment of psychoactive drugs and informs the development of strategies aimed at prevention and mitigation.

Flunitrazepam and its metabolites compromise zebrafish nervous system functionality: An integrated microbiome, metabolome, and genomic analysis.

The psychotropic drug flunitrazepam (FLZ) is frequently detected in aquatic environments, yet its neurotoxicity to aquatic organisms has not received sufficient attention. In this study, microbiome, metabolome, and genome analyses were conducted to study the effects of FLZ and its metabolite 7-aminoflunitrazepam (7-FLZ) on the zebrafish nervous system and understand their toxic mechanisms. The results demonstrated that drug exposure induced gut dysbiosis, decreased short-chain fatty acids and promoted the production of lipopolysaccharides (LPS). LPS entered the brain and interacted with Toll-like receptors to cause neuroinflammation by upregulating the expression of proinflammatory cytokines TNFα and NF-κB. The increased ratio of S-adenosylmethionine to S-adenosylhomocysteine in brain tissues indicated abnormal expression of Dnmt1 gene. Whole-genome bisulfite sequencing displayed an increase in differentially methylated regions (DMRs) associated-genes and pertinent biological pathways encompassed the MAPK signaling pathway, calcium signaling pathway, and Wnt signaling pathway. Correlation analysis confirmed connections between gut microbiota, their metabolites, inflammatory factors, and DNA methylation-related markers in brain tissue. These findings indicate that while the toxicity is somewhat reduced in metabolized products, both FLZ and 7-FLZ can induce DNA methylation in brain tissue and ultimately affect the biological function of the nervous system by disrupting gut microbiota and their metabolites.

Anxiety-like Behavior and GABAAR/BDZ Binding Site Response to Progesterone Withdrawal in a Stress-Vulnerable Strain, the Wistar Kyoto Rats.

Stress susceptibility could play a role in developing premenstrual anxiety due to abnormalities in the hypothalamus-pituitary-adrenal (HPA) axis and impairments in the GABAA receptors' benzodiazepine (BDZ) site. Hence, we studied the stress-vulnerable Wistar Kyoto rat strain (WKY) to evaluate progesterone withdrawal (PW) effects on anxiety, HPA axis response, and to explore indicators of GABAA functionality in the BDZ site. For five days, ovariectomized WKY rats were administered 2.0 mg/kg of progesterone. Twenty-four hours after the last administration, rats were tested in the anxiety-like burying behavior test (BBT) or elevated plus maze test (EPM), and corticosterone was determined. [3H]Flunitrazepam binding autoradiography served as the BDZ binding site index of the GABAA receptor in amygdala nuclei and hippocampus's dentate gyrus (DG). Finally, different doses of diazepam in PW-WKY rats were tested in the BBT. PW induced anxiety-like behaviors in both BBT and EPM compared with No-PW rats. PW increased corticosterone, but was blunted when combined with PW and BBT. PW increased [3H]Flunitrazepam binding in the DG and central amygdala compared with No-PW rats. Diazepam at a low dose induced an anxiogenic-like response in PW rats, suggesting a paradoxical response to benzodiazepines. Overall, PW induced anxiety-like behavior, a blunted HPA axis response, and higher GABAAR/BZD binding site sensitivity in a stress-vulnerable rat strain. These findings demonstrate the role of stress-susceptibility in GABAAR functionality in a preclinical approximation of PMDD.

High Fat Diet Increases [3H] Flunitrazepam Binding in the Mouse Brain that is Dependent on the Expression of the Dopamine D2 Gene.

Dopamine is an important neuromodulator in the brain that binds to dopamine D1-like receptors (D1, D5) as well as dopamine D2-like receptors (D2, D3, D4). The D2 receptor is known to play an integral role in a variety of physiological processes including addictive behaviors, locomotion, motivation, feeding behavior, and more. It was recently reported that dopamine is a direct-acting modulator of mammalian GABA(A) receptors. To this end, we wanted to examine how the expression of the dopamine D2 gene impacts the expression of GABA(A) receptors in the brain under different dietary conditions. Adult female Drd2 wild-type (WT), heterozygous (HT), and knockout (KO) mice were given either normal or high-fat diet for a period of 30 weeks. Following this, their brains were collected for [3H] Flunitrazepam binding in order to assess GABA(A) receptor expression. A high fat diet significantly increased [3H] Flunitrazepam binding in the regions of the somatosensory cortex, striatum, and various other cortical areas within WT mice. In contrast, no effect of diet was observed in HT or KO mice. As such, HT and KO mice displayed reduced [3H] Flunitrazepam binding in these areas relative to WT mice under high-fat dietary conditions. The effect of a high-fat diet on [3H] Flunitrazepam binding is consistent with recent evidence showing increases in GABA neurotransmitter levels following a high-fat diet. We demonstrate for the first time that the expression of the D2 gene plays a prominent role in the ability of a high-fat diet to impact GABA(A) receptors in the mouse brain.

Suppression of glutathione system and upregulation of caspase 3-dependent apoptosis mediate rohypnol-induced gastric injury.

Objectives: This study investigated the impact of rohypnol on gastric tissue integrity.Methods: Forty male Wistar rats were randomized into control, low dose rohypnol-treated, high dose rohypnol-treated, low dose rohypnol-treated recovery and high dose rohypnol-treated recovery groups.Results: Rohypnol caused significant rise in gastric malondialdehyde (MDA), oxidized glutathione (GSSG), nitric oxide (NO), tumour necrotic factor-α (TNF-α), and interleukin-6 (IL-6) levels. Also, rohypnol caused reductions in gastric reduced glutathione (GSH) (as well as GSH/GSSG), and activities of superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), glutathione peroxidase (GPx), cyclo-oxygenase (COX-2). Furthermore, rohypnol upregulated caspase 3 activity and induced gastric DNA damage, evident by a rise in 8-hydroxydeoxyguanosine (8-OHdG) and DNA fragmentation index (DFI) in gastric tissue. These alterations were coupled with reduced gastric weight and distorted gastric cytoarchitecture. Cessation of rohypnol caused a significant but not complete reversal of rohypnol-induced gastric damage.Conclusion: This study revealed that rohypnol induced gastric injury by suppressing glutathione content and COX-2 activity, and upregulating caspase 3-dependent apoptosis, which was partly reversed by rohypnol withdrawal.

Upregulation of Uric Acid Production and Caspase 3 Signalling Mediates Rohypnol-Induced Cardiorenal Damage.

The global prevalence of illicit drug use is on the increase with attendant complications like cardiorenal collapse. One such substance of abuse is rohypnol. Despite its ban in most countries, it remains a popular substance of abuse. Whether or not rohypnol induces cardiorenal injury and the associated mechanism is yet to be elucidated. Therefore, the present study investigated the effect of rohypnol on cardiorenal integrity and functions, and glucolipid metabolism. Forty-eight male Wistar rats randomized into six groups (n = 8/group) received (per os) vehicle, low-dose (2 mg/kg) and high-dose (4 mg/kg) rohypnol once daily for twenty eight days, with or without a cessation period. Data revealed that rohypnol exposure irreversibly caused insulin resistance, hyperglycaemia, and dyslipidaemia. This was accompanied by reduced cardiorenal mass and impaired cardiorenal cytoarchitecture and function. Furthermore, rohypnol treatment promoted oxidative stress, inflammation, genotoxicity, and decreased cardiorenal activities of Na+-K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase. These alterations were associated with enhanced uric acid generation and caspase 3 activity in the cardiorenal complex. Thus, this study reveals that rohypnol exposure triggers cardiorenal toxicity with incident insulin resistance, glucolipid and cardiorenal proton pump dysregulation, altered redox state, and inflammation via enhancement of uric acid generation and caspase 3-dependent mechanism.

Wearable Electronic Finger for Date Rape Drugs Screening: From "Do-It-Yourself" Fabrication to Self-Testing.

In-house digital fabrication of low-cost sensors that can on-site and rapidly detect adulteration of alcoholic beverages with sedation drugs (known as date rape drugs, (DRDs)) and analgesics is of great importance for everyday consumers and supervisory authorities. DRDs and analgesics are administrated in spirits for "drug-facilitated sexual assault" crimes and for the reduction of the following day hangover caused by low-quality spirits, respectively. This work describes, a novel "do-it-yourself" wearable 3D printed electrochemical finger (e-finger), which enables direct, rapid, and multianalyte self-testing of the main DRDs (flunitrazepam, scopolamine, ketamine) and paracetamol via direct immersing into a spirit shot. The oxygen interference on flunitrazepam detection was alleviated by dissolving an effervescent tablet of vitamin C in the spirit shot, as ascorbic acid serves as a scavenger for dissolved oxygen. The e-finger can be printed in-house at any size by anyone with access to a low-cost domestic 3D printer using a simple, fast, and low-cost printing procedure. The e-finger is addressed by a smartphone-based miniature potentiostat and allows on-the-spot self-checking of the quality and safety of alcoholic spirits, via a single calibration-free voltammetric measurement, readily performed even by untrained end users. The e-finger is a new powerful screening tool in the hands of supervisory authorities to conduct on-site forensic investigations. More importantly, it paves the way toward in-house e-production of "ready-to-use" reliable self-testing devices.

Long-Term Detection in Hair of Zolpidem, Oxazepam and Flunitrazepam in a Case of Drug-Facilitated Sexual Assault.

Drug-facilitated sexual assault (DFSA) cases are pretty common in forensic toxicology. In this case report, a 56-year-old female tourist claimed to have been sexually assaulted by five men after having had a drug-spiked alcoholic drink. Urine samples were collected at 38, 44 and 45 h after the alleged rape. After 7 months, hair strands (28 cm in length) were also sampled to perform the segmental hair testing. The urine samples and decontaminated hair segments were tested for different groups of basic, acidic and neutral substances (γ-hydroxybutyrate or GHB, Z-drugs, barbiturates, benzodiazepines, hypnotics, antipsychotics and drugs of abuse). Gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods were applied for the qualitative and quantitative analyses. Toxicological analyses performed on the urine samples showed inconclusive findings. Zolpidem, flunitrazepam and oxazepam were detected in the hair segments corresponding to the time frame of the alleged assault. The endogenous levels of GHB were detected along the hair shaft. No drugs were detected in the proximal and distal hair segments or in washing solutions. This DFSA case demonstrated that the segmental toxicological analysis of hair, even when performed 7 months after the sexual assault, can provide evidence consistent with a single exposure to psychoactive drugs, at the time of the offense.

Chronic neuroleptic treatment combined with a high fat diet elevated [3H] flunitrazepam binding in the cerebellum.

Clinical and preclinical studies have shown dysfunctions in genetic expression and neurotransmission of γ-Aminobutyric acid (GABA), GABAA receptor subunits, and GABA-synthesizing enzymes GAD67 and GAD65 in schizophrenia. It is well documented that there is significant weight gain after chronic neuroleptic treatment in humans. While there are limited studies on the effects of diet on GABA signaling directly, a change in diet has been used clinically as an adjunct to treatment for schizophrenic relief. In this study, rats chronically consumed either a chow diet (CD) or a 60% high-fat diet (HFD) and drank from bottles that contained one of the following solutions: water, haloperidol (1.5 mg/kg), or olanzapine (10 mg/kg) for four weeks. Rats were then euthanized and their brains were processed for GABAA in-vitro receptor autoradiography using [3H] flunitrazepam. A chronic HFD treatment yielded significantly increased [3H] flunitrazepam binding in the rat cerebellum independent of neuroleptic treatment. The desynchronization between the prefrontal cortex and the cerebellum is associated with major cognitive and motor dysfunctions commonly found in schizophrenic symptomatology, such as slowed reaction time, motor dyscoordination, and prefrontal activations related to speech fluency and cognitive alertness. These data support the notion that there is a dietary effect on GABA signaling within the cerebellum, as well as the importance of considering nutritional intervention methods as an adjunct treatment for patients chronically treated with neuroleptics. Finally, we indicate that future studies involving the analysis of individual patient's genetic profiles will further assist towards a precision medicine approach to the treatment of schizophrenia.

Sedative-Hypnotic Agents That Impact Gamma-Aminobutyric Acid Receptors: Focus on Flunitrazepam, Gamma-Hydroxybutyric Acid, Phenibut, and Selank.

There are many nonopioid central nervous system depressant substances that share a gamma-aminobutyric acid (GABA) receptor-related mechanism of action. These sedatives-hypnotics can be indicated to treat anxiety, seizures, depression, and insomnia but are also used as substances of abuse and used to facilitate sexual assault. Barbiturates, methaqualone, and glutethimide were among the first type A GABA receptor-mediated sedative-hypnotics. Their clinical use was limited for most indications by serious adverse events and strong abuse potential but continue to be used illicitly around the world. The benzodiazepines supplanted barbiturates for most indications because they were less likely to cause severe adverse events in monotherapy. Flunitrazepam is a newer benzodiazepine that is preferentially used recreationally and to facilitate sexual assault. Flunitrazepam has greater potency and higher affinity for the type A GABA receptor than most benzodiazepines. Gamma-hydroxybutyric acid is sought illicitly for its hypnotic, euphoric and anabolic effects as well as to facilitate sexual assault. When any of these GABAergic drugs are used in high doses or with other sedative hypnotic agents, respiratory depression, coma, and death have occurred. Chronic use of these GABAergic drugs can lead to significant withdrawal syndromes. Phenibut and selank are poorly studied Russian drugs with GABAergic mechanisms that are inexplicably sold to US consumers as dietary supplements. Poison control center calls regarding phenibut have increased substantially over the past 5 years. Desired euphoriant effects account for the recreational and illicit use of many GABA-modulating agents. However, illicit use can lead to significant toxicities related to abuse, dependence, and subsequent withdrawal syndromes. Significant evaluation of developing agents with GABA properties should be conducted to determine abuse potential before public access ensues.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes.

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023-0.043 pg/hair strand (0.077-0.36 pg/mg) for TZ, 0.11-0.63 pg/hair strand (0.44-5.2 pg/mg) for EZ, 0.14-2.6 pg/hair strand (0.56-22 pg/mg) for FNZ, 0.33-1.7 pg/hair strand (1.3-17 pg/mg) for NZ and 20-40 pg/hair strand (120-270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3-9.2 pg/hair strand (9.2-82 pg/mg) and 2.4-9.1 pg/hair strand (8.0-55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15-29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Binding activity of Valeriana fauriei root extract on GABAA receptor flunitrazepam sites and distribution of its active ingredients in the brain of mice - A comparison with that of V. officinalis root.

ETHNOPHARMACOLOGICAL RELEVANCE: Valeriana fauriei root (VF) is a crude drug registered in the Japanese Pharmacopeia 17th Edition and a known substitute for V. officinalis (VO). Although VO has been pharmacologically evaluated for its sedative effects and mechanism of action, data regarding VF remain scarce. AIM OF THE STUDY: We compared the binding affinity of VF and VO extracts, as well as examined the active ingredients in the VF extract, on flunitrazepam sites of γ-aminobutyric acid receptor type A (GABAA receptor). Furthermore, we confirmed whether these active ingredients were distributed in the brain of mice orally administered the VF extract. MATERIALS AND METHODS: We prepared the assay system to evaluate the binding activity of flunitrazepam sites of GABAA receptor using a 96-well plate and assessed the activities of VF and VO extracts. We then analyzed their constituents using HPLC with principal component analysis (PCA) and evaluated active ingredients correlated with their activities. The distribution of active ingredients in the plasma and brain of mice orally administered the VF extract prepared with different emulsifiers were analyzed by LC-MS/MS. RESULTS: The ethanol extract of VF exhibited significantly higher activity on flunitrazepam sites of GABAA receptor than VO. For the VF extract, kessyl glycol diacetate (KGD) was markedly associated with the binding activities; however, active ingredients included KGD, kessyl glycol 8-acetate (KG8), α-kessyl acetate (α-KA), and coniferyl isovalerate (CI). For VO, valerenic acid and five other compounds were associated with the binding affinity on flunitrazepam sites of GABAA receptor. On emulsifying the VF extract with a fat-soluble glycerin fatty acid ester, the plasma and brain distributions of KGD tended to be higher, those of KG8 were significantly more than 10-times higher, and those of α-KA was lower than those of the VF extract emulsified with water-soluble gum arabic, after oral administration in mice. CONCLUSIONS: Based on the binding activity on flunitrazepam sites of GABAA receptor and brain distribution, KGD, KG8, and α-KA can be considered active ingredients of VF. The addition of a fat-soluble emulsifier promoted the absorption of KGD, the main active ingredient, and KGD was metabolized to KG8 in the body. The present results suggest a possible mechanism underlying the sedative effect for VF, and these three compounds can be used as marker compounds to evaluate the quality of VF products.

All solid-state miniaturized potentiometric sensors for flunitrazepam determination in beverages.

Flunitrazepam is one of the frequently used hypnotic drugs to incapacitate victims for sexual assault. Appropriate diagnostic tools should be available to victims regarding the growing concern about "date-rape drugs" and their adverse impact on society. Miniaturized screen-printed potentiometric sensors offer crucial point-of-care devices that alleviate this serious problem. In this study, all solid-state screen-printed potentiometric flunitrazepam sensors have been designed. The paper device was printed with silver and carbon ink. Formation of an aqueous layer in the interface between carbon-conducting material and ion-sensing membrane nevertheless poses low reproducibility in the solid-contact electrodes. Accordingly, poly(3,4-ethylenedioxythiophene) (PEDT) nano-dispersion was applied as a conducting hydrophobic polymer on the electrode surface to curb water accumulation. Conditioning of ion-sensing membrane in the vicinity of reference membrane has been considered carefully using special protocol. Electrochemical characteristics of the proposed PEDT-based sensor were calculated and compared favorably to PEDT-free one. The miniaturized device was successfully used for the determination of flunitrazepam in carbonated soft drinks, energy drink, and malt beverage. Statistical comparison between the proposed sensor and official method revealed no significant difference. Nevertheless, the proposed sensor provides simple and user-friendly diagnostic tool with less equipment for on-site determination of flunitrazepam.

Rohypnol-induced sexual dysfunction is via suppression of hypothalamic-pituitary-testicular axis: An experimental study in rats.

Sexual activity is an essential part of reproductive functions and needed for the maintenance of fertility. Drugs, particularly substances of abuse, impair male reproductive function either by interrupting hormonal functions or through the nonhormonal pathways. This study evaluated the impact of Rohypnol use in sexual behaviour. Materials and methods: Thirty adult male Wistar rats of comparable weights (180-200 g) were randomly allocated into three groups, the control and low-dose and high-dose Rohypnol-treated groups. The control group received 0.5 ml of distilled water, while the low- and high-dose Rohypnol-treated groups received 2 mg/kg b.w and 4 mg/kg b.w of Rohypnol via oral lavage once daily for 28 days. Rohypnol significantly increased mount latency, intromission latency, ejaculation latency and post-ejaculatory interval, as well as lowered mount frequency, intromission frequency and ejaculation frequency. Rohypnol-induced sexual dysfunction was found to be associated with significant suppression of circulatory follicle-stimulating hormone, luteinising hormone, testosterone and oestrogen. The present study reveals that Rohypnol induces sexual dysfunction through suppression of hypothalamic-pituitary-testicular axis. It also implicates Rohypnol as a potential candidate for drug-induced infertility.

A Benzodiazepine Ligand with Improved GABAA Receptor α5-Subunit Selectivity Driven by Interactions with Loop C.

The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures, and many others. The most frequent GABAA receptor subtype is composed of two α-, two ß-, and one γ2-subunit, whereas the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5-containing GABAA receptors. Beyond the central nervous system, α5-containing GABAA receptors in airway smooth muscles are considered as an emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3 (SH53d-ester). Although SH53d-ester is only moderately selective for α5-subunit-containing GABAA receptors, the derivative SH53d-acid shows superior (>40-fold) affinity selectivity and is a positive modulator. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with human embryonic kidney 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5-subunit-selective drug candidate. SIGNIFICANCE STATEMENT: In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2'F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom-level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α-subunit is the dominant molecular determinant of drug selectivity.

Rapid Drop-Volume Electrochemical Detection of the "Date Rape" Drug Flunitrazepam in Spirits Using a Screen-Printed Sensor in a Dry-Reagent Format.

Flunitrazepam is an extremely potent benzodiazepine sedative which is associated with "drug-facilitated sexual assault" when administered within an alcoholic drink. This work describes a simple electrochemical method for on-site rapid detection of flunitrazepam in untreated spirits (whiskey, vodka and gin) using a single-use screen-printed sensor (featuring graphite working and auxiliary electrodes and an Ag/AgCl reference electrode) in a dry reagent format. Analysis was performed by placing a drop of sample on the sensor, which was previously coated with dry KCl, and recording selected reduction/oxidation peaks of the target compound in a cyclic voltammetry scan. The limit of quantification of flunitrazepam was at the sub-mg L-1 range. The between-sensor % relative standard deviation of the analytically useful reduction peak in a solution containing 11.4 mg L-1 flunitrazepam was 9.8% (n = 5). Quantification was performed using calibration curves constructed from pooled samples spiked with flunitrazepam with relative errors <15%. The main advantages of the methodology are that it involves no sample pretreatment (such as deoxygenation, extraction or reagent(s) addition) and requires only drop-sized volumes of the sample, thus facilitating rapid on-site screening using portable equipment.

A noble electrochemical sensor based on TiO2@CuO-N-rGO and poly (L-cysteine) nanocomposite applicable for trace analysis of flunitrazepam.

Flunitrazepam or date rape medication with trade name of Rohypnol belongs to the benzodiazepines branch that is used as a sedative, anesthetic, anticonvulsant, muscle relaxant, and antianxiety drug. It is known as "drug of aggression" because of its very strong and long-lasting effects on the central nervous system. The sedative influence of flunitrazepam drug increases with alcohol drinking, which causes mental and motor disorders and causes the victim to become silent. Due to its criminals use, its accurate measurement is crucial. In this work, a novel electrochemical sensor based on TiO2@CuO-N doped rGO, TiO2@CuO-N-rGO, nano-composite and poly (L-cysteine), poly (L-Cys), is presented for trace analysis of flunitrazepam in aqueous solution. At first, TiO2@CuO-N-rGO nano-composite was synthesized by the sol-gel method and characterized by Raman spectroscopy, Fourier transform infrared, field emission scanning electron microscope, and X-ray diffraction analysis. Then, the suspension of the TiO2@CuO-N-rGO nano-composite was drop casted on the surface of the glassy carbon electrode (GCE/TiO2@CuO-N-rGO). After that, electro-polymerization of l-cysteine on the GCE/TiO2@CuO-N-rGO surface was performed by cyclic voltammetry (CV) method. The electrochemical characteristics of the GCE/TiO2@CuO-N-rGO/poly (L-Cys) surface were evaluated in the solution of ferri/ferrocyanide by electrochemical impedance spectroscopy (EIS) and CV techniques. The increase in current, change in oxidation peak potential, and the appearance of two reduction peaks indicated higher electron transfer rate with well-performed electrochemical process of flunitrazepam at the modified electrode surface compared to the bare GCE. These improvements originate from the synergistic effect of TiO2@CuO-N-rGO nano-composite and poly (L-Cys). Finally, a linear relationship was resulted between the oxidation peak current and the concentration of flunitrazepam in the wide concentration range of 1 nM to 50 µM with a detection limit of 0.3 nM.

Hops compounds modulatory effects and 6-prenylnaringenin dual mode of action on GABAA receptors.

Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABAA receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABAA receptors. Flumazenil sensitivity of GABA-potentiating effects, [3H]Ro 15-4513, and [3H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner where the IC50 values for this potentiation in native GABAA receptors were 3.2 µM and 3.7 µM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [3H]EBOB binding. [3H]Ro 15-4513 and [3H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABAA receptor α1ß2γ2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABAA receptors where it may act as a positive allosteric modulator at α+ß- binding interface as well as a null modulator at the flumazenil-sensitive α+γ2- binding interface.