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.

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.

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.

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.

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.

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.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS).

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.

Delayed creatine supplementation counteracts reduction of GABAergic function and protects against seizures susceptibility after traumatic brain injury in rats.

Traumatic brain injury (TBI) is a devastating disease frequently followed by behavioral disabilities including post-traumatic epilepsy (PTE). Although reasonable progress in understanding its pathophysiology has been made, treatment of PTE is still limited. Several studies have shown the neuroprotective effect of creatine in different models of brain pathology, but its effects on PTE is not elucidated. Thus, we decided to investigate the impact of delayed and chronic creatine supplementation on susceptibility to epileptic seizures evoked by pentylenetetrazol (PTZ) after TBI. Our experimental data revealed that 4 weeks of creatine supplementation (300 mg/kg, p.o.) initiated 1 week after fluid percussion injury (FPI) notably increased the latency to first myoclonic and tonic-clonic seizures, decreased the time spent in tonic-clonic seizure, seizure intensity, epileptiform discharges and spindle oscillations induced by a sub-convulsant dose of PTZ (35 mg/kg, i.p.). Interestingly, this protective effect persists for 1 week even when creatine supplementation is discontinued. The anticonvulsant effect of creatine was associated with its ability to reduce cell loss including the number of parvalbumin positive (PARV+) cells in CA3 region of the hippocampus. Furthermore, creatine supplementation also protected against the reduction of GAD67 levels, GAD activity and specific [3H]flunitrazepam binding in the hippocampus. These findings showed that chronic creatine supplementation may play a neuroprotective role on brain excitability by controlling the GABAergic function after TBI, providing a possible new strategy for the treatment of PTE.

Cerebral Malaria Causes Enduring Behavioral and Molecular Changes in Mice Brain Without Causing Gross Histopathological Damage.

Malaria, parasitic disease considered a major health public problem, is caused by Plasmodium protozoan genus and transmitted by the bite of infected female Anopheles mosquito genus. Cerebral malaria (CM) is the most severe presentation of malaria, caused by P. falciparum and responsible for high mortality and enduring development of cognitive deficits which may persist even after cure and cessation of therapy. In the present study we evaluated selected behavioral, neurochemical and neuropathologic parameters after rescue from experimental cerebral malaria caused by P. berghei ANKA in C57BL/6 mice. Behavioral tests showed impaired nest building activity as well as increased marble burying, indicating that natural behavior of mice remains altered even after cure of infection. Regarding the neurochemical data, we found decreased α2/α3 Na+,K+-ATPase activity and increased immunoreactivity of phosphorylated Na+,K+-ATPase at Ser943 in cerebral cortex after CM. In addition, [3H]-Flunitrazepam binding assays revealed a decrease of benzodiazepine/GABAA receptor binding sites in infected animals. Moreover, in hippocampus, dot blot analysis revealed increased levels of protein carbonyls, suggesting occurrence of oxidative damage to proteins. Interestingly, no changes in the neuropathological markers Fluoro-Jade C, Timm staining or IBA-1 were detected. Altogether, present data indicate that behavioral and neurochemical alterations persist even after parasitemia clearance and CM recovery, which agrees with available clinical findings. Some of the molecular mechanisms reported in the present study may underlie the behavioral changes and increased seizure susceptibility that persist after recovery from CM and may help in the future development of therapeutic strategies for CM sequelae.

Metabolic Profile of Flunitrazepam: Clinical and Forensic Toxicological Aspects.

BACKGROUND: Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat insomnia and as a pre-anesthetic agent. The illicit practice in drug-facilitated sexual assault led to important clinical and forensic concerns. OBJECTIVE: In this work the metabolism of FNZ, and pharmacological- and toxicological-related effects, were fully reviewed. METHODS: FNZ and related known metabolizing enzymes and metabolites were searched in books and in PubMed (U.S. National Library of Medicine) without a limiting period. RESULTS: Major metabolic pathways include N-demethylation, 3-hydroxylation, nitro-reduction, and further N-acetylation of the amino group, yielding N-desmethylflunitrazepam, 3-hydroxy-flunitrazepam, 7-aminoflunitrazepam, and 7-acetamidoflunitrazepam, respectively. A combination of these reactions may lead to the formation of 7-amino-N-desmethylflunitrazepam, 7-acetamido-N-desmethylflunitrazepam, 3- hydroxy-7-aminoflunitrazepam, 3-hydroxy-7-acetamidoflunitrazepam, 3-hydroxy-N-desmethylflunitrazepam and glucuronide conjugates. Genotypic variations in enzymes, interactions with other drugs or stability of FNZ during storage can result in large interindividual variability in the toxicological results. CONCLUSION: It is aimed that knowing the metabolism of FNZ may lead to the development of new analytical strategies for early detection, since this drug is typically present in very low concentrations in blood and urine when used to facilitate sexual assault.

Effects of acute and chronic administration of neurosteroid dehydroepiandrosterone sulfate on neuronal excitability in mice.

BACKGROUND: Neurosteroid dehydroepiandrosterone sulfate (DHEAS) has been associated with important brain functions, including neuronal survival, memory, and behavior, showing therapeutic potential in various neuropsychiatric and cognitive disorders. However, the antagonistic effects of DHEAS on γ-amino-butyric acidA receptors and its facilitatory action on glutamatergic neurotransmission might lead to enhanced brain excitability and seizures and thus limit DHEAS therapeutic applications. The aim of this study was to investigate possible age and sex differences in the neuronal excitability of the mice following acute and chronic DHEAS administration. METHODS: DHEAS was administered intraperitoneally in male and female adult and old mice either acutely or repeatedly once daily for 4 weeks in a 10 mg/kg dose. To investigate the potential proconvulsant properties of DHEAS, we studied the effects of acute and chronic DHEAS treatment on picrotoxin-, pentylentetrazole-, and N-methyl-D-aspartate-induced seizures in mice. The effects of acute and chronic DHEAS administration on the locomotor activity, motor coordination, and body weight of the mice were also studied. We also investigated the effects of DHEAS treatment on [(3)H]flunitrazepam binding to the mouse brain membranes. RESULTS: DHEAS did not modify the locomotor activity, motor coordination, body weight, and brain [(3)H]flunitrazepam binding of male and female mice. The results failed to demonstrate significant effects of single- and long-term DHEAS treatment on the convulsive susceptibility in both adult and aged mice of both sexes. However, small but significant changes regarding sex differences in the susceptibility to seizures were observed following DHEAS administration to mice. CONCLUSION: Although our findings suggest that DHEAS treatment might be safe for various potential therapeutic applications in adult as well as in old age, they also support subtle interaction of DHEAS with male and female hormonal status, which may underline observed sex differences in the relationship between DHEAS and various health outcomes.

Discovery of a novel general anesthetic chemotype using high-throughput screening.

BACKGROUND: The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. METHODS: Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. RESULTS: From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. CONCLUSION: The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

Synaptosomal membrane-based Langmuir-Blodgett films: a platform for studies on γ-aminobutyric acid type A receptor binding properties.

In this work we used Langmuir-Blodgett films (LB) as model membranes to study the effect of molecular packing on the flunitrazepam (FNZ) accessibility to the binding sites at the GABAA receptor (GABAA-R). Ligand binding data were correlated with film topography analysis by atomic force microscopy images (AFM) and SDS-PAGE. Langmuir films (LF) were prepared by the spreading of synaptosomal membranes (SM) from bovine brain cortex at the air-water interface. LBs were obtained by the transference, at 15 or 35 mN/m constant surface pressure (π), of one (LB15/1c and LB35/1c) or two (LB35/2c) LFs to a film-free hydrophobic alkylated substrate (CONglass). Transference was performed in a serial manner, which allowed the accumulation of a great number of samples. SDS-PAGE clearly showed a 55 kDa band characteristic of GABAA-R subunits. Detrended fluctuation analysis of topographic data from AFM images exhibited a single slope value (self-similarity parameter α) in CONglass and a discontinuous slope change in the α value at an autocorrelation length of ∼100 nm in all LB samples, supporting the LF transference to the substrate. AFM images of CONglass and LB15/1c exhibited roughness and average heights that were similar between measurements and significantly lower than those of LB35/1c and LB35/2c, suggesting that the substrate coverage in the latter was more stable than in LB15/1c. While [(3)H]FNZ binding in LB15/1c did not reach saturation, in LB35/1c the binding kinetics became sigmoid with a binding affinity lower than in the SM suspension. Our results highlight the π dependence of both binding and topological data and call to mind the receptor mechanosensitivity. Thus, LB films provide a tool for bionanosensing GABAA-R ligand binding as well as GABAA-R activity modulation induced by the environmental supramolecular organization.

Effect of synthetic steroids on GABAA receptor binding in rat brain.

Neuroactive steroids, like allopregnanolone (A) and pregnanolone (P), bind to specifics sites on the GABAA receptor complex and modulate receptor function. They are capable to inhibit or stimulate the binding of GABAA receptor-specific ligands, like t-butyl-bicyclophosphorothionate, flunitrazepam and muscimol. We have previously characterized a set of oxygen-bridged synthetic steroids (SS) analogs to A or P using synaptosomes. Considering that the subunit composition of the GABAA receptor throughout the central nervous system affects the magnitude of the modulation of the GABAA receptor by NAS, we evaluated the action of two selected SS, in brain sections containing the cerebral cortex (CC) and hippocampus (HC) using quantitative receptor autoradiography. Both SS affected the binding of the three ligands in a similar way to A and P, with some differences on certain CC layers according to the ligand used. One of the SS, the 3α-hydroxy-6,19-epoxypregn-4-ene-20-one (compound 5), behaved similarly to the natural neuroactive steroids. However, significant differences with compound 5 were observed on the HC CA2 region, making it steroid suitable for a specific action. Those differences may be related to structural conformation of the SS and the subunits' composition present on the receptor complex.

Production of human metabolites by gastrointestinal bacteria as a potential source of post-mortem alteration of antemortem drug/metabolite concentrations.

Previous studies have demonstrated that bacterial species are capable of transforming complex chemical substances. Several of these species, native to the human gastrointestinal tract, are active in postmortem decomposition. They have potential to cause biotransformations affecting compound-to-metabolite ratios within the human body, especially after death. Investigation of postmortem effects could supply valuable information, especially concerning compound identification and confirmation. The purpose of this research was to investigate the effects of Escherichia coli, Bacteroides fragilis, and Clostridium perfringens on diazepam and flunitrazepam in Reinforced Clostridial Medium, and to compare bacterial biotransformation products to those of human metabolism. A decrease in diazepam concentration between pre- and post-incubation was observed for samples inoculated with Escherichia coli (14.7-20.2%) as well as Bacteroides fragilis (13.9-25.7%); however there was no corresponding increase in concentration for the monitored human metabolites. Flunitrazepam demonstrated a greater concentration loss when incubated with individual bacterial species as well as mixed culture (79.2-100.0%). Samples incubated with Bacteroides fragilis, Clostridium perfringens, and mixed culture resulted in nearly complete conversion of flunitrazepam. Increased 7-aminoflunitrazepam concentrations accounted for the majority of the conversion; however discrepancies in the mass balance of the reaction suggested the possibility of a minor metabolite that was not monitored in the current analysis. These experiments served as a pilot study and proof of concept that can be adapted and applied to a realm of possibilities. Ultimately, this methodology would be ideal to study compounds that are too toxic or lethal for animal and human metabolic investigations.

Temporal characteristics of stress-induced decrease in benzodiazepine reception in C57BL/6 and BALB/c mice.

We studied the duration of the drop of specific (3)H-flunitrazepam binding by synaptosomal membranes from the brain of C57Bl/6 and BALB/c mice after open-field and "contact with predator" tests. It was found that reduced benzodiazepine reception in BALB/c mice after open-field test persisted for 1.5 h, but no changes of this parameter were found in C57Bl/6 mice. After contact with predator, the binding capacity of the benzodiazepine site of GABAA receptor was reduced for 8 h in BALB/c mice and for 24 h in C57Bl/6 mice.

[Brain benzodiazepine receptors in humans and rats with alcohol addiction].

OBJECTIVE: Benzodiazepine receptors (BDR) in synaptosomal and mitochondrial membranes from different brain areas of alcohol abused patients (postmortem) and the brain cortex of male rats (Vistar line) with different preference to alcohol were studied. METHODS: Synaptosomal and mitochondrial receptors of membranes from different brain areas of patients with alcohol addiction and controls were explored using radioreceptor analysis with selective ligands [3H]flunitrazepam and [3H]PK-11195. BDR in the rat brain were studied using [3H]flunitrazepam and [3H]Ro5-4864. RESULTS: An analysis of kinetic parameters (Kd and Bmax) of [3H]flunitrazepam and [3H]PK-11195 binding with synaptosomal and mitochondrial membranes in the human brain showed that BDR was decreased and capacity increased in different human brain areas under the influence of alcohol abuse. The most distinct changes were found in the prefrontal cortex, nucleus caudatus and cerebella cortex. Alcohol abuse induced greater changes in mitochondrial membranes compared to synaptosomal membranes that was consistent with physiological and defensive functions of mitochondrial membranes and CNS under the influence of toxic substances. The affinity of [3H]flunitrazepam and [3H]Ro5-4864 binding with membranes was decreased, but the capacity of receptors was increased in the brain cortex of alcohol-preferring male rats compared to alcohol non-preferring rats. Administration of anticonvulsant meta-chloro-benzhydryl-urea to rats prefer ethanol increased the affinity of BDR.

Inhibitory effects of carvone isomers on the GABAA receptor in primary cultures of rat cortical neurons.

Carvone is a natural terpene which can be purified as R-(-) or S-(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA(A) receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA(A) by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA-induced stimulation of [(3)H]flunitrazepam binding, suggesting their interaction with the GABA(A) receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R-(-)-carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA(A) receptor at its noncompetitive blocker site.

Effect of meta-chlorobenzhydryl urea (m-ClBHU) on benzodiazepine receptor system in rat brain during experimental alcoholism.

Chronic alcohol intake induces neuroadaptive changes in benzodiazepine receptors modulating GABAA receptors that promote alcohol addiction. Analysis of benzodiazepine receptors in the brain of Wistar rats differing by alcohol preference has demonstrated that affinity of [(3)H]flunitrazepam and [(3)H]Ro5-4864 binding with membrane fraction was reduced, while the density of specific binding sites in the brain cortex of heavy drinking and low drinking rats was increased in comparison with rats nonpreferring alcohol. Administration of anticonvulsant meta-chlorobenzhydryl urea increased affinity of benzodiazepine receptors in the brain cortex of heavy drinking rats, which improved GABA neurotransmission in the brain of these animals and reduced alcohol consumption.