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.

Notas de farmacología: estudios de disolución de medicamentos del sistema nervioso central / Pharmacology notes: drug dissolution studies of the central nervous system

El propósito de los estudios in vitro es comparar las características de liberación del principio activo contenido en una forma farmacéutica sólida oral mediante la cantidad, o porcentaje de la dosis, disuelta en función del tiempo en condiciones controladas y validadas. Se realizó una comparación de disolución in vitro de flunitrazepam (Rohypnol®, Somnidual® e Inervon®) y de lamotrigina (Lamictal® y Epilepax®). Para los perfiles de disolución de flunitrazepam, una de las marcas comerciales tuvo un perfil de disolución muy diferente a las otras dos marcas. Para el caso de lamotriginano existió diferencia para aquellos pH que se consideran fundamentales para la disolución del comprimido en el tracto gastrointestinal. Los resultados obtenidos de los estudios in vitro son simplemente orientadores, permiten tan solo guiar la puesta en marcha del ensayo de bioequivalencia entre el test evaluado in vitro y la referencia utilizada en el mismo ensayo. No obstante, estos resultados pueden constituirse en un elemento de apoyo a la presunción de bioinequivalencia como causa de un evento adverso (ineficacia o toxicidad) detectado en los programas de farmacovigilancia actualmente en marcha.

A synthetic bioisoster of trimethadione and phenytoin elicits anticonvulsant effect, protects the brain oxidative damage produced by seizures and exerts antidepressant action in mice.

Epilepsy is recognized as one of the most common and serious neurological disorder affecting 1-2% of the world׳s population. The present study demonstrates that systemic administration of 3-butyl-5,5-dimethyl-1,2,3-oxathiazolidine-4-one-2,2-dioxide (DIOXIDE), a synthetic compound bioisoster of trimethadione and phenytoin (classical anticonvulsants), elicits a dose dependent anticonvulsant response in mice submitted to the subcutaneous pentylenetetrazole seizure test (scPTZ). Among various factors supposed to play role in epilepsy, oxidative stress and reactive species have strongly emerged. The protection exerted by DIOXIDE over the extent of brain oxidative damage produced by PTZ was determined, by measuring the levels of lipid peroxidation and reduced glutathione and the activity of Na(+)/K(+)-ATPase. Psychiatric disorders represent frequent comorbidities in persons with epilepsy. In this report, the potential anxiolytic and antidepressant activities of DIOXIDE were evaluated in several widely used models for assessing anxiolytic and antidepressant activities in rodents. Although DIOXIDE did not evidence anxiolytic activity at the doses tested, it revealed a significant antidepressant-like effect. Preliminary studies of its mechanism of action, by means of its capacity to act via the GABAA receptor (using the [(3)H]flunitrazepam binding assay in vitro and the picrotoxin test in vivo) and the Na(+) channel (using the alkaloid veratrine, a voltage-Na(+) channel agonist) demonstrated that the anticonvulsant effect is not likely related to the GABAergic pathway and the antidepressant-like effect could be due to its Na(+) channel blocking properties. The results for DIOXIDE suggested it as a new anticonvulsant-antioxidant and antidepressant compound that deserves further development.

Physical exercise improves synaptic dysfunction and recovers the loss of survival factors in 3xTg-AD mouse brain.

Physical exercise has become a potentially beneficial therapy for reducing neurodegeneration symptoms in Alzheimer's disease. Previous studies have shown that cognitive deterioration, anxiety and the startle response observed in 7-month-old 3xTg-AD mice were ameliorated after 6 months of free access to a running wheel. Also, alterations in synaptic response to paired-pulse stimulation were improved. The present study further investigated some molecular mechanisms underlying the beneficial effects of 6 months of voluntary exercise on synaptic plasticity in 7-month-old 3xTg-AD mice. Changes in binding parameters of [(3)H]-flunitrazepam to GABAA receptor and of [(3)H]-MK-801 to NMDA receptor in cerebral cortex of 3xTgAD mice were restored by voluntary exercise. In addition, reduced expression levels of NMDA receptor NR2B subunit were reestablished. The synaptic proteins synaptophysin and PSD-95 and the neuroprotective proteins GDNF and SIRT1 were downregulated in 3xTgAD mice and were recovered by exercise treatment. Overall, in this paper we highlight the fact that different interrelated mechanisms are involved in the beneficial effects of exercise on synaptic plasticity alterations in the 3xTg-AD mouse model.

GABA-induced uncoupling of GABA/benzodiazepine site interactions is mediated by increased GABAA receptor internalization and associated with a change in subunit composition.

Persistent activation of GABAA receptors triggers compensatory changes in receptor function that are relevant to physiological, pathological and pharmacological conditions. Chronic treatment of cultured neurons with GABA for 48h has been shown to produce a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine site interactions with a half-time of 24-25h. Down-regulation is the result of a transcriptional repression of GABAA receptor subunit genes and depends on activation of L-type voltage-gated calcium channels. The mechanism of this uncoupling is currently unknown. We have previously demonstrated that a single brief exposure of rat primary neocortical cultures to GABA for 5-10min (t½=3min) initiates a process that results in uncoupling hours later (t½=12h) without a change in receptor number. Uncoupling is contingent upon GABAA receptor activation and independent of voltage-gated calcium influx. This process is accompanied by a selective decrease in subunit mRNA levels. Here, we report that the brief GABA exposure induces a decrease in the percentage of α3-containing receptors, a receptor subtype that exhibits a high degree of coupling between GABA and benzodiazepine binding sites. Initiation of GABA-induced uncoupling is prevented by co-incubation of GABA with high concentrations of sucrose suggesting that it is dependent on a receptor internalization step. Moreover, results from immunocytochemical and biochemical experiments indicate that GABA exposure causes an increase in GABAA receptor endocytosis. Together, these data suggest that the uncoupling mechanism involves an initial increase in receptor internalization followed by activation of a signaling cascade that leads to selective changes in receptor subunit levels. These changes might result in the assembly of receptors with altered subunit compositions that display a lower degree of coupling between GABA and benzodiazepine sites. Uncoupling might represent a homeostatic mechanism that negatively regulates GABAergic transmission under physiological conditions in which synaptic GABAA receptors are transiently activated for several minutes.

Anxiolytic effects of Julibroside C1 isolated from Albizzia julibrissin in mice.

Julibroside C1 is a saponin-containing compound isolated from Albizzia julibrissin Durazz. In this study, we investigated the putative anxiolytic effects of Julibroside C1 using the elevated plus maze (EPM) in mice. Julibroside C1 at doses of 0.5 and 1 mg/kg significantly increased the time spent in the open arms and the number of entries into the open arms of the EPM compared to the control group. Moreover, the anxiolytic-like effects of Julibroside C1 (0.5 mg/kg) were blocked by WAY-100635 (5-HT1A receptor antagonist), bicuculline (GABA(A) receptor antagonist), and flumazenil (antagonist of the GABA(A) receptor benzodiazepine site). However, Julibroside C1 did not change locomotor activity or induce myorelaxant effects. We used quantitative receptor autoradiography to investigate the effects of Julibroside C1 on alterations in mouse brain receptors. After acute treatment with Julibroside C1 (0.5 mg/kg), [(3)H]-8-OH-DPAT binding was significantly decreased in the CA1 region of the hippocampus and [(3)H]-flunitrazepam binding was decreased remarkably in the cingulate cortex region. However, [(3)H]-muscimol binding did not show a significant change in any brain region. Taken together, our findings suggest that Julibroside C1 shows anxiolytic-like effects, which might be mediated by the 5-HT1A and GABA(A)-benzodiazepine receptor systems.

A rapid UPLC-MS/MS method for simultaneous determination of flunitrazepam, 7-aminoflunitrazepam, methadone and EDDP in human, rat and rabbit plasma.

A simple, high-throughput, sensitive LC-ESI-MS/MS method is presented for the simultaneous determination of methadone (MET), flunitrazepam (FNZ) and their major metabolites, EDDP (2-ethilidene-1,5-dimethyl-3,3-diphenylpyrrolidone) and 7-aminoflunitrazepam (7-AFNZ), respectively, in human, rat and rabbit plasma. The isolation of the selected compounds involved a liquid-liquid extraction with ethyl acetate at a basic pH. Good chromatographic separation was achieved on a HSS T3 column (1.8 µm particle size), with a 3 min gradient elution using a mixture of acetonitrile with 0.1% formic acid (solvent A) and 5mM ammonium acetate (solvent B) as the mobile phase. The tandem mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with ionization of the analytes in positive mode. The assay was fully validated according to current acceptance criteria for bioanalytical methods validation. It was proved to be linear in the range of 0.5-250 ng/mL, with adequate accuracy and precision over this range. Based on accuracy and CV% values the LOQ and ULOQ values were set at 0.509 ng/mL and 2036 ng/mL for MET, 0.520 ng/mL and 2080 ng/mL for EDDP, 0.524 ng/mL and 2096 ng/mL for FNZ and 0.528 ng/mL and 2114 ng/mL for 7-AFNZ, respectively. The method was tested for potential matrix effects, without observing significant ion suppression. The investigated compounds stability was examined in plasma at room temperature and after three freeze-thaw cycles and in the final extract when maintained at 4 °C in the autosampler. Potential stability issues were observed only for FNZ at room temperature. The method was successfully applied to quantify the selected compounds in human, rat and rabbit plasma samples, after exposure to FNZ or simultaneous exposure to FNZ and MET.

Rats with different thresholds for DMCM-induced clonic convulsions differ in the sleep-time of diazepam and [(3)H]-Ro 15-4513 binding.

The current study investigated the possible inherent relationship between convulsions and sleep involving the GABA(A)/benzodiazepine site complex. The aim of this study was to determine if rats with high (HTR) and low (LTR) thresholds for clonic convulsions induced by DMCM, a benzodiazepine inverse agonist, differ in the following aspects: (1) sensitivity to the hypnotic effects of the GABA(A) positive allosteric modulators diazepam, pentobarbital and ethanol and (2) in the binding of [(3)H]-flunitrazepam, a benzodiazepine agonist, measured by autoradiography, and [(3)H]-Ro 15-4513, a benzodiazepine partial inverse agonist, to membranes from discrete brain regions. The LTR subgroup presented a shorter diazepam-induced sleeping time compared to that of the HTR subgroup. Biochemical assays revealed that the LTR subgroup did not differ in [(3)H]-flunitrazepam binding compared to the HTR subgroup. With respect to the binding of [(3)H]-Ro 15-4513, the LTR subgroup had higher binding in the brainstem and lower binding in the striatum compared to the HTR subgroup. These results suggest that differences in the benzodiazepine site on the GABA(A) receptor may underlie the susceptibility to DMCM-induced convulsions and sensitivity to the hypnotic effect of diazepam.

Urinary detection times and excretion patterns of flunitrazepam and its metabolites after a single oral dose.

We investigated the excretion profiles of flunitrazepam metabolites in urine after a single dose. Sixteen volunteers received either 0.5 or 2.0 mg flunitrazepam. Urine samples were collected after 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 240, and 336 h. Samples were screened using CEDIA (300 microg/L cutoff) and quantitated using liquid chromatography-tandem mass spectrometry. The cutoff was 0.5 microg/L for flunitrazepam, N-desmethylflunitrazepam, 7-aminoflunitrazepam, 7-aminodesmethylflunitrazepam, 7-acetamidoflunitrazepam, and 7-acetamidodesmethylflunitrazepam. None of the subjects receiving 0.5 mg were screened positive, and only 23 of 102 samples from the subjects given 2.0 mg were positive with CEDIA. The predominant metabolites were 7-aminoflunitrazepam and 7-aminodesmethylflunitrazepam. For all subjects given the low dose, 7-aminoflunitrazepam was detected up to 120 h, and for two subjects for more than 240 h. Seven subjects given the high dose were positive up to 240 h for 7-aminoflunitrazepam. We conclude that the ratio 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam increased with time, independent of dose, and may be used to estimate the time of intake. For some low-dose subjects, the metabolite concentrations in the early samples were low and a chromatographic method may fail to detect the intake. We think laboratories should consider this when advising police and hospitals about sampling as well as when they set up strategies for analysis.

Decreased GABAA receptors and benzodiazepine binding sites in the anterior cingulate cortex in autism.

The anterior cingulate cortex (ACC; BA 24) via its extensive limbic and high order association cortical connectivity to prefrontal cortex is a key part of an important circuitry participating in executive function, affect, and socio-emotional behavior. Multiple lines of evidence, including genetic and imaging studies, suggest that the ACC and gamma-amino-butyric acid (GABA) system may be affected in autism. The benzodiazepine binding site on the GABA(A) receptor complex is an important target for pharmacotherapy and has important clinical implications. The present multiple-concentration ligand-binding study utilized (3)H-muscimol and (3)H-flunitrazepam to determine the number (B(max)), binding affinity (K(d)), and distribution of GABA(A) receptors and benzodiazepine binding sites, respectively, in the ACC in adult autistic and control cases. Compared to controls, the autistic group had significant decreases in the mean density of GABA(A) receptors in the supragranular (46.8%) and infragranular (20.2%) layers of the ACC and in the density of benzodiazepine binding sites in the supragranular (28.9%) and infragranular (16.4%) lamina [corrected]. These findings suggest that in the autistic group this downregulation of both benzodiazepine sites and GABA(A) receptors in the ACC may be the result of increased GABA innervation and/or release disturbing the delicate excitation/inhibition balance of principal neurons as well as their output to key limbic cortical targets. Such disturbances likely underlie the core alterations in socio-emotional behaviors in autism.

[Abuse of alcohol and benzodiazepine during substitution therapy in heroin addicts: a review of the literature]. / Abus d'alcool et de benzodiazépines lors des traitements de substitution chez l'héroïnomane : une revue de la littérature.

INTRODUCTION: In spite of its seriousness, dependence on alcohol and benzodiazepines during substitution treatment are poorly documented. Its frequency is nonetheless significant. According to studies, between one and two thirds of patients are affected. This consumption is under verbalized by patients and underestimated by carers. In one study, where the average diazepam doses were from 40 to 45 mg per day, 30% of the patients were taking 70 to 300 mg per day, two thirds having experimented with a fixed dose of 100mg. Benzodiazepines, especially diazepam and flunitrazepam, were studied versus placebo. Thus, 10 to 20mg of diazepam gave rise to euphoria, a sensation of being drugged, sedation and lessening of cognitive performance. The aim of this consumption is to potentiate the euphoria induced by opioids, a "boost" effect during the hour after taking it, or the calming of the outward signs of withdrawal. The most sought after molecules are the most sedative, those with pronounced plasmatic peaks, and the most accessible. LITERATURE FINDINGS: In multidependant subjects, opioid dependence had been earlier in adolescence, with a number of therapeutic failures. They had been faced with repetitive rejection and separation during childhood, medicolegal and social problems. Somatization, depression, anxiety and psychotic disorders are frequent in this subgroup. Heavy drinkers under methadone treatment are highly vulnerable to cocaine. Their behaviour is at risk, with exchange of syringes; their survival rate is 10 years less than that of moderate consumers of alcohol. Most are single, with a previous prison, psychiatric or addictive cursus and they present significant psychological vulnerability. For some authors, benzodiazepines indicate a psychiatric comorbidity. Methadone significantly reduces the consumption of alcohol by nonalcoholic heroin addicts. Although alcohol is an enzymatic inductor of methadone catabolism, with bell-shaped methadone plasma curves over 24 hours, a substitution treatment is recommended. It has a minimum impact on care, in spite of efficiency and retention in therapeutical programs, allowing the subject's inclusion in the framework of a more regular and sustained medical follow-up. Treatment of benzodiazepine dependence by a progressive regression of doses has little efficacy in subjects which cannot control how much medication they are taking. Certain authors have suggested maintenance treatments of clonezepam. The most appropriate therapeutic propositions are: (1) maintenance of therapeutic links though a framework of deliverance from flexible substitution treatment; (2) prevention by cautious prescribing and control of dispensing medication; (3) parallel treatment of psychiatric comorbidities and related personality disorders; (4) individual psychiatric treatment, either institutional or in consistent networks.

Buprenorphine alters desmethylflunitrazepam disposition and flunitrazepam toxicity in rats.

High-dosage buprenorphine (BUP) consumed concomitantly with benzodiazepines (BZDs) including flunitrazepam (FZ) may cause life-threatening respiratory depression despite a BUP ceiling effect and BZDs' limited effects on ventilation. However, the mechanism of BUP/FZ interaction remains unknown. We hypothesized that BUP may alter the disposition of FZ active metabolites in vivo, contributing to respiratory toxicity. Plasma FZ, desmethylflunitrazepam (DMFZ), and 7-aminoflunitrazepam (7-AFZ) concentrations were measured using gas chromatography-mass spectrometry. Intravenous BUP 30 mg/kg pretreatment did not alter plasma FZ and 7-AFZ kinetics in Sprague-Dawley rats infused with 40 mg/kg FZ over 30 min, whereas resulting in a three-fold increase in the area under the curve (AUC) of DMFZ concentrations compared with control (p < 0.01). In contrast, BUP did not significantly modify plasma DMFZ concentrations after intravenous infusion of 7 mg/kg DMFZ, whereas resulting in a similar peak concentration to that generated from 40 mg/kg FZ administration. Regarding the effects on ventilation, BUP (30 mg/kg) as well as its combination with FZ (0.3 mg/kg) significantly increased PaCO(2), whereas only BUP/FZ combination decreased PaO(2) (p < 0.001). Interestingly, FZ (40 mg/kg) but not DMFZ (40 mg/kg) significantly increased PaCO(2) (p < 0.05), whereas DMFZ but not FZ decreased PaO(2) (p < 0.05). Thus, decrease in PaO(2) appears related to BUP-mediated effects on DMFZ disposition, although increases in PaCO(2) relate to direct BUP/FZ additive or synergistic dynamic interactions. We conclude that combined high-dosage BUP and FZ is responsible for increased respiratory toxicity in which BUP-mediated alteration in DMFZ disposition may play a significant role.

The role of transcriptional and translational mechanisms in flumazenil-induced up-regulation of recombinant GABA(A) receptors.

The aim of this study was to further elucidate the mechanisms involved in adaptive changes of GABA(A) receptors following prolonged exposure to flumazenil, the antagonist of benzodiazepine binding sites on GABA(A) receptors. The effects of prolonged flumazenil treatment were studied on recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors stably expressed in human embryonic kidney (HEK 293) cells. Using radioligand binding experiments we found enhancement in the maximum number of [(3)H]muscimol labeled binding sites in different preparations of HEK 293 cells. The parallel increase of [(3)H]flunitrazepam binding sites in the membranes was reduced in the presence of actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, respectively. Chronic flumazenil also raised the steady-state level of mRNA encoding alpha(1) receptor subunit. The results suggest that the up-regulation of GABA(A) receptors, observed after prolonged flumazenil treatment is at least partly due to increased de novo synthesis of receptor proteins at both transcriptional and translational level.

Prediction of metabolic clearance using fresh human hepatocytes: comparison with cryopreserved hepatocytes and hepatic microsomes for five benzodiazepines.

1. Predictions of in vivo intrinsic clearance from cryopreserved human hepatocytes may be systematically low. In the current study, the metabolite kinetics of a series of CYP3A4 substrates (benzodiazepines) in fresh human hepatocytes from five donors, via a major UK supplier, were investigated and compared with those previously reported (by the authors' laboratory) for cryopreserved human hepatocytes and hepatic microsomes. 2. A high incidence of autoactivation (up to tenfold) and heteroactivation (by testosterone, up to 14-fold) among the major pathways was observed. CYP capacity (V(max)) was marginally lower and 'affinity' constants (K(M), S(50)) were marginally greater compared with cryopreserved hepatocytes. 3. Average intrinsic clearance (based on maximal clearance, CL(max)) was sevenfold lower than in cryopreserved hepatocytes (reflecting sensitivity of intrinsic clearance estimation in vitro to mechanistic parameter values, particularly those involving atypical kinetics), but scaled intrinsic clearances for fresh (and cryopreserved) hepatocytes were within the range previously determined in hepatic microsomes. 4. There was no evidence from this series of studies that fresh hepatocytes provide quantitatively improved estimates of intrinsic clearance over cryopreserved hepatocytes.

Modulation of human risky decision making by flunitrazepam.

RATIONALE: GABA-modulating drugs produce disinhibitory effects that increase the probability of risk-taking behavior. Previous reports suggest that the misuse of the benzodiazepine flunitrazepam is associated with several forms of harmful risky behavior, including theft, violence, and intoxication-related auto accidents. OBJECTIVES: The present study examined the dose-response relationships between acute flunitrazepam administration and human decision making under conditions of risk. The analyses also examined flunitrazepam-mediated changes in decision-making processes using a computational modeling approach, the expectancy valence model (EVM). MATERIALS AND METHODS: Using a laboratory measure of risky decision making designed to address acute drug effects, 12 adults were administered placebo, 0.5, 1.0, and 2.0 mg/70 kg flunitrazepam in a within-subject, repeated measures counterbalanced design. Flunitrazepam was compounded and doses were administered in an 8-oz liquid solution. Primary data analyses examined subjective effects, response rates, distribution of choices between the risky and nonrisky option, and personality correlates related to peak drug effects. Individual-subject data were submitted to a computational modeling analysis (EVM) that provided parameter estimates corresponding to components of valence; updating expectancies about alternatives (learning/memory); and consistency between choices and expected outcomes (sensitivity to learned outcomes). RESULTS: Flunitrazepam produced dose-related changes in subjective effects and response rates, and increased selection of the risky response option. High doses significantly changed decision-making processes related to the learning/memory and consistency parameters. CONCLUSIONS: At sufficiently high doses, flunitrazepam can engender increases in risky decision making. Globally, these changes appear similar to previous effects we have observed after acute administration of alcohol and alprazolam. As suggested by the EVM outcomes, the mechanisms underlying the changes in risky decision making are more similar to alprazolam than alcohol.

[Comparative study of drug efficacy and drug additives between generic drugs and original drugs].

In the present study, we tested three kinds of sleeping drugs, consisting mainly of triazolam, brotizolam, and flunitrazepam, to compare the drug efficacy of generic drugs with that of original drugs. After these drugs were administered orally to mice, drug efficacy was evaluated in terms of ambulation, onset time of sleep, and duration of sleep in the open field test. For all kinds of sleep-inducing drugs, the drug efficacy of most generic drugs is not necessarily equal to that of the original drug. The main reason for the difference appears to be due to differences in the rate of absorption of the main drug. Any other differences between an original drug and a generic drug are caused by drug additives, the crystal form of the main drug, the formulation, and so on. In this study, the formulation was not the reason for the differences because all of the drugs were pulverized in a mortar and had no special coating. The drug additives for all the drugs are listed and the drug efficacy compared. Unfortunately, the information was not sufficient to shed any light on the differences in drug efficacy. For effective drug therapy, more information on drug additives should be provided.

Relative abuse liability of gamma-hydroxybutyric acid, flunitrazepam, and ethanol in club drug users.

OBJECTIVES: Despite the increasing concern about gamma-hydroxybutyric acid (GHB) toxicity, there are few studies examining the clinical pharmacology of GHB and its abuse potential. To evaluate GHB-induced subjective and physiological effects, its relative abuse liability and its impact on psychomotor performance in club drug users. MATERIALS AND METHODS: Twelve healthy male recreational users of GHB participated in 5 experimental sessions in the framework of a clinical trial. The study was randomized, double-blind, double-dummy, and crossover. Drug conditions were a single oral dose of GHB (40 or 60 mg/kg), ethanol (0.7 g/kg), flunitrazepam (1.25 mg), and placebo. Study variables included vital signs (blood pressure, heart rate, oral temperature, pupil diameter), psychomotor performance (digit symbol substitution test, balance, Maddox-Wing), subjective effects (a set of 13 visual analogue scales, Addiction Research Center Inventory-49 items, and Evaluation of the Subjective Effects of Substances with Potential of Abuse questionnaires), and pharmacokinetics. RESULTS: All active conditions induced positive effects related to their abuse potential. The administration of GHB produced euphoria and pleasurable effects with slightly higher ratings than those observed for flunitrazepam and ethanol. Gamma-hydroxybutyric acid induced a biphasic time profile with an initial stimulant-like effect related to the simultaneous rise of plasma concentrations and a latter sedative effect not related to GHB kinetics. Gamma-hydroxybutyric acid increased blood pressure and pupil diameter. Ethanol induced its prototypical effects, and flunitrazepam produced marked sedation. Gamma-hydroxybutyric acid and flunitrazepam impaired psychomotor performance, digit symbol substitution test, and balance task, whereas ethanol, at the dose tested, induced only mild effects exclusively affecting the balance task. CONCLUSIONS: Our results suggest a high abuse liability of GHB and flunitrazepam in club drug users.

Postnatal development and kinetics of [3H]gaboxadol binding in rat brain: in vitro homogenate binding and quantitative autoradiography.

The postnatal development of the binding of the GABA(A) receptor agonist [(3)H]gaboxadol in rat brain was investigated. Using brain tissue from rats obtained at postnatal days 1, 10, 25, and >25 (adult), the binding of [(3)H]gaboxadol and the benzodiazepine [(3)H]flunitrazepam to GABA(A) receptors was compared in homogenate binding assays and quantitative receptor autoradiography. Kinetic and equilibrium data obtained in homogenate binding studies revealed two different [(3)H]gaboxadol affinities. A kinetically derived K(D) of 3.7 nM in adult cerebellum, calculated from the association and dissociation rate constants k(on) (1.45 x 10(8) M(-1) min(-1)) and k(off) (0.54 min(-1)) was contrasted by an equilibrium K(D) of 38.6 nM, obtained by homologous competition experiments. Quantitative analysis of autoradiographic data revealed an increase in specific [(3)H]gaboxadol binding sites during brain development, which resembles the anatomical and temporal pattern of the postnatal expression of the extrasynaptic delta subunit of GABA(A) receptors. In conclusion, by the radioligand binding data obtained on native tissue, binding of gaboxadol to GABA(A) receptors located outside the synaptic junctions could be postulated.

Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex.

Rat forebrain synaptosomes were extracted with Triton X-100 at 4 degrees C and the insoluble material, which is enriched in post-synaptic densities (PSDs), was subjected to sedimentation on a continuous sucrose gradient. Two pools of Triton X-100-insoluble gamma-aminobutyric acid type-A receptors (GABA(A)Rs) were identified: (i) a higher-density pool (rho = 1.10-1.15 mg/mL) of GABA(A)Rs that contains the gamma2 subunit (plus alpha and beta subunits) and that is associated to gephyrin and the GABAergic post-synaptic complex and (ii) a lower-density pool (rho = 1.06-1.09 mg/mL) of GABA(A)Rs associated to detergent-resistant membranes (DRMs) that contain alpha and beta subunits but not the gamma2 subunit. Some of these GABA(A)Rs contain the delta subunit. Two pools of GABA(A)Rs insoluble in Triton X-100 at 4 degrees C were also identified in cultured hippocampal neurons: (i) a GABA(A)R pool that forms clusters that co-localize with gephyrin and remains Triton X-100-insoluble after cholesterol depletion and (ii) a GABA(A)R pool that is diffusely distributed at the neuronal surface that can be induced to form GABA(A)R clusters by capping with an anti-alpha1 GABA(A)R subunit antibody and that becomes solubilized in Triton X-100 at 4 degrees C after cholesterol depletion. Thus, there is a pool of GABA(A)Rs associated to lipid rafts that is non-synaptic and that has a subunit composition different from that of the synaptic GABA(A)Rs. Some of the lipid raft-associated GABA(A)Rs might be involved in tonic inhibition.