Brain PET imaging using 11C-flumazenil and 11C-buprenorphine does not support the hypothesis of a mutual interaction between buprenorphine and benzodiazepines at the neuroreceptor level.

Among opioids, buprenorphine presents a favorable safety profile with a limited risk of respiratory depression. However, fatalities have been reported when buprenorphine is combined to a benzodiazepine. Potentiation of buprenorphine interaction with opioid receptors (ORs) with benzodiazepines, and/or vice versa, is hypothesized to explain this drug-drug interaction (DDI). The mutual DDI between buprenorphine and benzodiazepines was investigated at the neuroreceptor level in nonhuman primates (n = 4 individuals) using brain PET imaging and kinetic modelling. The binding potential (BPND) of benzodiazepine receptor (BzR) was assessed using 11C-flumazenil PET imaging before and after administration of buprenorphine (0.2 mg, i.v.). Moreover, the brain kinetics and receptor binding of buprenorphine were investigated in the same individuals using 11C-buprenorphine PET imaging before and after administration of diazepam (10 mg, i.v.). Outcome parameters were compared using a two-way ANOVA. Buprenorphine did not impact the plasma nor brain kinetics of 11C-flumazenil. 11C-flumazenil BPND was unchanged following buprenorphine exposure, in any brain region (p > 0.05). Similarly, diazepam did not impact the plasma or brain kinetics of 11C-buprenorphine. 11C-buprenorphine volume of distribution (VT) was unchanged following diazepam exposure, in any brain region (p > 0.05). To conclude, our PET imaging findings do not support a neuropharmacokinetic or neuroreceptor-related mechanism of the buprenorphine/benzodiazepine interaction.

Mechanisms of respiratory depression induced by the combination of buprenorphine and diazepam in rats.

BACKGROUND: The safety profile of buprenorphine has encouraged its widespread use. However, fatalities have been attributed to benzodiazepine/buprenorphine combinations, by poorly understood mechanisms of toxicity. Mechanistic hypotheses include (i) benzodiazepine-mediated increase in brain buprenorphine (pharmacokinetic hypothesis); (ii) benzodiazepine-mediated potentiation of buprenorphine interaction with opioid receptors (receptor hypothesis); and (iii) combined effects of buprenorphine and benzodiazepine on respiratory parameters (pharmacodynamic hypothesis). METHODS: We studied the neuro-respiratory effects of buprenorphine (30 mg kg-1, i.p.), diazepam (20 mg kg-1, s.c.), and diazepam/buprenorphine combination in rats using arterial blood gas analysis, plethysmography, and diaphragm electromyography. Pretreatments with various opioid and gamma-aminobutyric acid receptor antagonists were tested. Diazepam impact on brain 11C-buprenorphine kinetics and binding to opioid receptors was studied using positron emission tomography imaging. RESULTS: In contrast to diazepam and buprenorphine alone, diazepam/buprenorphine induced early-onset sedation (P<0.05) and respiratory depression (P<0.001). Diazepam did not alter 11C-buprenorphine brain kinetics or binding to opioid receptors. Diazepam/buprenorphine-induced effects on inspiratory time were additive, driven by buprenorphine (P<0.0001) and were blocked by naloxonazine (P<0.01). Diazepam/buprenorphine-induced effects on expiratory time were non-additive (P<0.001), different from buprenorphine-induced effects (P<0.05) and were blocked by flumazenil (P<0.01). Diazepam/buprenorphine-induced effects on tidal volume were non-additive (P<0.01), different from diazepam- (P<0.05) and buprenorphine-induced effects (P<0.0001) and were blocked by naloxonazine (P<0.05) and flumazenil (P<0.05). Compared with buprenorphine, diazepam/buprenorphine decreased diaphragm contraction amplitude (P<0.01). CONCLUSIONS: Pharmacodynamic parameters and antagonist pretreatments indicate that diazepam/buprenorphine-induced respiratory depression results from a pharmacodynamic interaction between both drugs on ventilatory parameters.

A local-neighborhood Lassen plot filter for creating occupancy and non-displaceable binding images.

For radioligands without a reference region, the Lassen plot can be used to estimate receptor occupancy by an exogenous drug (ODrug). However, the Lassen plot is not well-suited for spatial variation in ODrug. To overcome this limitation, we introduce a Lassen plot filter, i.e. a Lassen plot applied to local neighborhoods in PET images. Image data were simulated with regional variation in VND, ODrug, both, or neither and analyzed using the change in binding potential (ΔBPND), the conventional Lassen plot, and the Lassen plot filter at the region of interest (ROI) and voxel level. All methods were also applied to a human [11C]flumazenil occupancy study using PF-06372865. This combination of a non-selective radioligand and selective drug should lead to varying ODrug provided the distribution of subtypes varies spatially. In contrast with ΔBPND and the conventional Lassen plot, ROI-level and voxel-level Lassen plot filter estimates remained unbiased in the presence of regional variation in VND or ODrug. In the [11C]flumazenil data-set, ODrug was shown to vary regionally in accordance with the distribution of binding sites for [11C]flumazenil and PF-06372865. We demonstrate that a local-neighborhood Lassen plot filter provides robust and unbiased estimates of ODrug and VND without the need for any user intervention.

Bolus infusion scheme for the adjustment of steady state [11C]Flumazenil levels in the grey matter and in the blood plasma for neuroreceptor imaging.

The use of hybrid PET/MR imaging facilitates the simultaneous investigation of challenge-related changes in ligand binding to neuroreceptors using PET, while concurrently measuring neuroactivation or blood flow with MRI. Having attained a steady state of the PET radiotracer using a bolus-infusion protocol, it is possible to observe alterations in ligand neuroreceptor binding through changes in distribution volumes. Here, we present an iterative procedure for establishing an administration scheme to obtain steady state [11C]flumazenil concentrations in grey matter in the human brain. In order to achieve a steady state in the shortest possible time, the bolus infusion ratio from a previous examination was adapted to fit the subsequent examination. 17 male volunteers were included in the study. Boli and infusions with different weightings were given to the subjects and were characterised by kbol values from 74 â€‹min down to 42 â€‹min. Metabolite analysis was used to ascertain the value of unmetabolised flumazenil in the plasma, and PET imaging was used to assess its binding in the grey matter. The flumazenil time-activity curves (TACs) in the brain were decomposed into activity contributions from pure grey and white matter and analysed for 12 â€‹vol of interest (VOIs). The curves highlighted a large variability in metabolic rates between the subjects, with kbol â€‹= â€‹54.3 â€‹min being a reliable value to provide flumazenil equilibrium conditions in the majority of the VOIs and cases. The distribution volume of flumazenil in all 12 VOIs was determined.

Altered cerebral benzodiazepine receptor binding in post-traumatic stress disorder.

Agonists of the γ-aminobutyric acid (GABA) type A benzodiazepine (BZD) receptor exert anxiolytic effects in anxiety disorders, raising the possibility that altered GABA-ergic function may play a role in the pathophysiology of anxiety disorders, such as post-traumatic stress disorder (PTSD). However, few neuroimaging studies have assessed the function or binding potential of the central GABAA BZD receptor system in PTSD. Therefore, our aim was to compare the BZD receptor binding potential between PTSD patients and healthy controls. Twelve medication-free participants with a current diagnosis of PTSD and 15 matched healthy controls underwent positron emission tomography (PET) imaging using [11C] flumazenil. Structural magnetic resonance imaging (MRI) scans were obtained and co-registered to the PET images to permit co-location of neuroanatomical structures in the lower resolution PET image data. Compared to healthy controls, PTSD patients exhibited increased BZD binding in the caudal anterior cingulate cortex and precuneus (p's < 0.05). Severity of PTSD symptoms positively correlated with BZD binding in the left mid- and anterior insular cortices. This study extends previous findings by suggesting that central BZD receptor system involvement in PTSD includes portions of the default mode and salience networks, along with insular regions that support interoception and autonomic arousal.

Preclinical comparison study between [18F]fluoromethyl-PBR28 and its deuterated analog in a rat model of neuroinflammation.

We designed and synthesized deuterium-substituted [18F]fluoromethyl-PBR28 ([18F]1-d2) as a novel translocator protein 18 kDa (TSPO)-targeted radioligand with enhanced in vivo stability. The comparison studies between [18F]fluoromethyl-PBR28 ([18F]1) and its deuterate analog ([18F]1-d2) were investigated in terms of in vitro binding affinity, lipophilicity and in vivo stability. In addition, the accuracies of both radioligands were determined by comparing the PET imaging data in the same LPS-induced neuroinflammation rat model. Both aryloxyanilide analogs showed similar lipophilicity and in vitro affinity for TSPO. However, [18F]1-d2 provided significantly lower femur uptake than [18F]1 (1.5 ±â€¯1.2 vs. 4.1 ±â€¯1.7%ID/g at 2 h post-injection) in an ex vivo biodistribution study. [18F]1-d2 was also selectively accumulated in the inflammatory lesion with the binding potential of the specifically bound radioligand relative to the non-displaceable radioligand in tissue (BPND = 3.17 ±â€¯0.48), in a LPS-induced acute neuroinflammation rat model, comparable to that of [18F]1 (BPND = 2.13 ±â€¯0.51). These results indicate that [18F]1-d2 had higher in vivo stability, which resulted in an enhanced target-to-background ratio compared to that induced by [18F]1.

Loss of inhibition in sensorimotor networks in focal hand dystonia.

OBJECTIVE: To investigate GABA-ergic receptor density and associated brain functional and grey matter changes in focal hand dystonia (FHD). METHODS: 18 patients with FHD of the right hand and 18 age and gender matched healthy volunteers (HV) participated in this study. We measured the density of GABA-A receptors using [11C] Flumazenil and perfusion using [15O] H2O. Anatomical images were also used to measure grey matter volume with voxel-based morphometry (VBM). RESULTS: In FHD patients compared to HV, the vermis VI of the right cerebellum and the left sensorimotor cortex had a decrease of Flumazenil binding potential (FMZ-BP), whereas the striatum and the lateral cerebellum did not show significant change. Bilateral inferior prefrontal cortex had increased FMZ-BP and an increase of perfusion, which correlated negatively with disease duration. Only the left sensorimotor cortex showed a decrease of grey matter volume. INTERPRETATION: Impairments of GABAergic neurotransmission in the cerebellum and the sensorimotor cortical areas could explain different aspects of loss of inhibitory control in FHD, the former being involved in maladaptive plasticity, the latter in surround inhibition. Reorganization of the inferior prefrontal cortices, part of the associative network, might be compensatory for the loss of inhibitory control in sensorimotor circuits. These findings suggest that cerebellar and cerebral GABAergic abnormalities could play a role in the functional imbalance of striato-cerebello-cortical loops in dystonia.

Parametric Imaging of [11C]Flumazenil Binding in the Rat Brain.

PURPOSE: This study evaluates the performance of several parametric methods for assessing [11C]flumazenil binding distribution in the rat brain. PROCEDURES: Dynamic (60 min) positron emission tomography data with metabolite-corrected plasma input function were retrospectively analyzed (male Wistar rats, n = 10). Distribution volume (V T) images were generated from basis function method (BFM), Logan graphical analysis (Logan), and spectral analysis (SA). Using the pons as pseudo-reference tissue, binding potential (BP ND and DVR-1) images were obtained from receptor parametric imaging algorithms (RPM and SRTM2) and reference Logan (RLogan). Standardized uptake value images (SUV and SUVR) were also computed for different intervals post-injection. Next, regional averages were extracted from the parametric images, using pre-defined volumes of interest, which were also applied to the regional time-activity curves from the dynamic data. Parametric data were compared to their regional counterparts and to two-tissue compartment model (2TCM)-based values (previously defined as the model of choice for rats). Parameter agreement was assessed by linear regression analysis and Bland-Altman plots. RESULTS: All parametric methods strongly correlated to their regional counterparts (R 2 > 0.97) and to the 2TCM values (R 2 ≥ 0.95). SA and RLogan underestimated V T and BP ND (slope of 0.93 and 0.86, respectively), while SUVR-1 overestimated BP ND (slope higher than 1.07 for all intervals). While BFM and SRTM2 had the smallest bias to 2TCM values (0.05 for both), ratio Bland-Altman plots showed Logan and RLogan displayed relative errors which were comparable between different regions, in contrast with the other methods. Although SUV consistently underestimated V T, the bias in this method was also constant across regions. CONCLUSIONS: All parametric methods performed well for the analysis of [11C]flumazenil distribution and binding in the rat brain. However, Logan and RLogan slightly outperformed the other methods in terms of precision, providing robust parameter estimation and constant bias. Yet, other methods can be of interest, because they can provide tissue perfusion (i.e., K 1 with BFM and SA), relative flow (i.e., R 1 with RPM and SRTM2), and model order (SA) images.

Contribution of neuroinflammation to changes in [11C]flumazenil binding in the rat brain: Evaluation of the inflamed pons as reference tissue.

INTRODUCTION: [11C]Flumazenil is a well-known PET tracer for GABAA receptors and is mainly used as an imaging biomarker for neuronal loss. Recently, GABAA receptors on immune cells have been investigated as target for modulation of inflammation. Since neuronal loss is often accompanied by neuroinflammation, PET imaging with [11C]flumazenil is potentially affected by infiltrating immune cells. This may also compromise the validity of using the pons as reference tissue in quantitative pharmacokinetic analysis. This study aims to evaluate whether inflammatory processes in the brain can influence [11C]flumazenil uptake and affect the outcome of pharmacokinetic modeling when the pons is used as reference tissue. METHODS: The herpes simplex encephalitis (HSE) rat model is known to cause neuroinflammation in the brainstem. Dynamic [11C]flumazenil PET scans of 60-min, accompanied by arterial blood sampling and metabolite analysis, were acquired at day 6-7days post-infection of male Wistar rats (HSE, n=5 and control, n=6). Additionally, the GABAA receptor was saturated by injection of unlabeled flumazenil prior to the tracer injection in 4 rats per group. PET data were analyzed by pharmacokinetic modeling. RESULTS: No statistically significant differences were found in the volume of distribution (VT) or non-displaceable binding potential (BPND) between control and HSE rats in any of the brain regions. Pre-saturation with unlabeled flumazenil resulted in a statistically significant reduction in [11C]flumazenil VT in all brain regions. The BPND obtained from SRTM exhibited a good correlation to DVR - 1 values from the two-tissue compartment model, coupled with some level of underestimation. CONCLUSION: Reliable quantification of [11C]flumazenil binding in rats can be obtained by pharmacokinetic analysis using the pons as a pseudo-reference tissue even in the presence of strong acute neuroinflammation.

Altered GABAA receptor density and unaltered blood-brain barrier [11C]flumazenil transport in drug-resistant epilepsy patients with mesial temporal sclerosis.

Studies in rodents suggest that flumazenil is a P-glycoprotein substrate at the blood-brain barrier. This study aimed to assess whether [11C]flumazenil is a P-glycoprotein substrate in humans and to what extent increased P-glycoprotein function in epilepsy may confound interpretation of clinical [11C]flumazenil studies used to assess gamma-aminobutyric acid A receptors. Nine drug-resistant patients with epilepsy and mesial temporal sclerosis were scanned twice using [11C]flumazenil before and after partial P-glycoprotein blockade with tariquidar. Volume of distribution, nondisplaceable binding potential, and the ratio of rate constants of [11C]flumazenil transport across the blood-brain barrier (K1/k2) were derived for whole brain and several regions. All parameters were compared between pre- and post-tariquidar scans. Regional results were compared between mesial temporal sclerosis and contralateral sides. Tariquidar significantly increased global K1/k2 (+23%) and volume of distribution (+10%), but not nondisplaceable binding potential. At the mesial temporal sclerosis side volume of distribution and nondisplaceable binding potential were lower in hippocampus (both ∼-19%) and amygdala (both ∼-16%), but K1/k2 did not differ, suggesting that only regional gamma-aminobutyric acid A receptor density is altered in epilepsy. In conclusion, although [11C]flumazenil appears to be a (weak) P-glycoprotein substrate in humans, this does not seem to affect its role as a tracer for assessing gamma-aminobutyric acid A receptor density.

GABA deficiency in NF1: A multimodal [11C]-flumazenil and spectroscopy study.

OBJECTIVE: To provide a comprehensive investigation of the γ-aminobutyric acid (GABA) system in patients with neurofibromatosis type 1 (NF1) that allows understanding the nature of the GABA imbalance in humans at pre- and postsynaptic levels. METHODS: In this cross-sectional study, we employed multimodal imaging and spectroscopy measures to investigate GABA type A (GABAA) receptor binding, using [(11)C]-flumazenil PET, and GABA concentration, using magnetic resonance spectroscopy (MRS). Fourteen adult patients with NF1 and 13 matched controls were included in the study. MRS was performed in the occipital cortex and in a frontal region centered in the functionally localized frontal eye fields. PET and MRS acquisitions were performed in the same day. RESULTS: Patients with NF1 have reduced concentration of GABA+ in the occipital cortex (p = 0.004) and frontal eye fields (p = 0.026). PET results showed decreased binding of GABAA receptors in patients in the parieto-occipital cortex, midbrain, and thalamus, which are not explained by decreased gray matter levels. CONCLUSIONS: Abnormalities in the GABA system in NF1 involve both GABA concentration and GABAA receptor density suggestive of neurodevelopmental synaptopathy with both pre- and postsynaptic involvement.

Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose.

BACKGOUND/AIM: Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2:1 ratio) applied as concomitant therapy. METHODS: Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ionmonitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column usingthe mixture of acidic acetonitrile and 20 mM of ammonium acetatein water (55 : 45) as a mobile phase. RESULTS: The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the sameextractant in the process of liquid-liquid extraction. The limit ofdetection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patientstreated with both flumazenil and aminophylline, the eliminationconstant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in.the patients who received flumazenil alone. CONCLUSION: The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.

Design of Infusion Schemes for Neuroreceptor Imaging: Application to [(11)C]Flumazenil-PET Steady-State Study.

This study aims at developing a simulation system that predicts the optimal study design for attaining tracer steady-state conditions in brain and blood rapidly. Tracer kinetics was determined from bolus studies and used to construct the system. Subsequently, the system was used to design inputs for bolus infusion (BI) or programmed infusion (PI) experiments. Steady-state quantitative measurements can be made with one short scan and venous blood samples. The GABAA receptor ligand [(11)C]Flumazenil (FMZ) was chosen for this purpose, as it lacks a suitable reference region. Methods. Five bolus [(11)C]FMZ-PET scans were conducted, based on which population-based PI and BI schemes were designed and tested in five additional healthy subjects. The design of a PI was assisted by an offline feedback controller. Results. The system could reproduce the measurements in blood and brain. With PI, [(11)C]FMZ steady state was attained within 40 min, which was 8 min earlier than the optimal BI (B/I ratio = 55 min). Conclusions. The system can design both BI and PI schemes to attain steady state rapidly. For example, subjects can be [(11)C]FMZ-PET scanned after 40 min of tracer infusion for 40 min with venous sampling and a straight-forward quantification. This simulation toolbox is available for other PET-tracers.

A microPET comparison of the effects of etifoxine and diazepam on [(11)C]flumazenil uptake in rat brains.

Using positron emission tomography (PET), the present study assessed the binding of [(11)C]flumazenil to GABA-A receptors in anesthetized rats following a single intravenous injection of an active dose of either etifoxine (25mg/kg) or diazepam (1mg/kg), which are both anxiolytic drugs. [(11)C]flumazenil binding was measured in five discrete brain structures, namely the caudate putamen, hippocampus, cerebellum, occipital cortex and parietal cortex. As expected, diazepam injection produced a significant decrease in [(11)C]flumazenil binding, which was interpreted as benzodiazepine GABA-A receptor occupancy, whereas etifoxine increased the binding of [(11)C]flumazenil. This first use of in vivo imaging after etifoxine administration revealed the activated binding pattern of [(11)C]flumazenil and highlighted the pharmacological differences between etifoxine and benzodiazepines. Using the same [(11)C]flumazenil radiotracer, PET neuroimaging could be applied to larger animals and, ultimately, to human subjects, thus providing new perspectives for better defining the molecular pharmacology of etifoxine.

Quantitative pharmacological analyses of the interaction between flumazenil and midazolam in monkeys discriminating midazolam: Determination of the functional half life of flumazenil.

The duration of action of a drug is commonly estimated using plasma concentration, which is not always practical to obtain or an accurate estimate of functional half life. For example, flumazenil is used clinically to reverse the effects of benzodiazepines like midazolam; however, its elimination can be altered by other drugs, including some benzodiazepines, thereby altering its half life. This study used Schild analyses to characterize antagonism of midazolam by flumazenil and determine the functional half life of flumazenil. Four monkeys discriminated 0.178mg/kg midazolam while responding under a fixed-ratio 10 schedule of stimulus-shock termination; flumazenil was given at various times before determination of a midazolam dose-effect curve. There was a time-related decrease in the magnitude of shift of the midazolam dose-effect curve as the interval between flumazenil and midazolam increased. The potency of flumazenil, estimated by apparent pA2 values (95% CI), was 7.30 (7.12, 7.49), 7.17 (7.03, 7.31), 6.91 (6.72, 7.10) and 6.80 (6.67, 6.92) at 15, 30, 60 and 120min after flumazenil administration, respectively. The functional half life of flumazenil, derived from potency estimates, was 57±13min. Thus, increasing the interval between flumazenil and midazolam causes orderly decreases in flumazenil potency; however, across a broad range of conditions, the qualitative nature of the interaction does not change, as indicated by slopes of Schild plots at all time points that are not different from unity. Differences in potency of flumazenil are therefore due to elimination of flumazenil and not due to pharmacodynamic changes over time.

Sedative and anxiolytic effects of the extracts of the leaves of Stachytarpheta cayennensis in mice.

The leaves are used ethnomedicinally in Nigeria and other parts of the world for insomnia and anxiety among other uses. The investigations sought scientific evidence for the ethnomedicinal use of the leaves for the management of insomnia and anxiety as well as the neural mechanisms for the activities. The sedative and anxiolytic effects of the extracts of the leaves of Stachytarpheta cayennensis were examined in this study. The methanolic extract (5-50 mg/kg, i.p.) as well as the ethylacetate (10-50 mg/kg, i.p.), butanol and aqueous fractions (5-50 mg/kg, i.p.) of the extract were examined. Sedation was assessed as reduced novelty-induced rearing (NIR), reduced spontaneous locomotor activity (SLA) and increased pentobarbitone-induced sleeping time (PIST) in mice. The anti-anxiety effect (methanol 2.5-5.0; butanol 5.0; aqueous 20.0; ethylacetate 25.0 mg/kg, i.p.) was assessed using an elevated plus maze. LD50 was calculated for the extract and the fractions after the intraperitoneal route of administration using the Locke method. The methanolic extract, the butanol and the aqueous fractions inhibited rearing and spontaneous locomotion but prolonged pentobarbitone induced sleep. The ethylacetate fraction however increased both rearing and locomotion and decreased pentobarbitone sleeping time. The butanol and aqueous fractions, but not the methanol extract showed indices of open arm avoidance consistent with anti-anxiety effect. Naltrexone (2.5 mg/kg, i.p.) reversed the inhibition of rearing, locomotion and prolongation of pentobarbitone sleep due to the aqueous fraction of the extract. Flumazenil (2mg/kg, i.p.) abolished the effects of both methanolic extract and the butanol fraction on rearing, locomotion, pentobarbitone sleep and anxiety model. The methanolic extract, the butanol and aqueous fractions possess sedative activity while the ethylacetate fraction possesses stimulant property. The anxiolytic effect was found in both the aqueous fraction and the butanol fraction but not in the main methanol extract and also not in the ethylacetate fraction. Flumazenil, blocked the effect of the leaves of Stachytarpheta cayennensis on rearing, locomotion and elevated plus maze suggesting that GABA receptors are involved in the observed sedative and anxiolytic activities. This study also found opioid receptors involved in the sedative activity of the leaves of Stachytarpheta cayennensis. The rationale for the ethnomedicinal use of the leaves for the management of insomnia and anxiety were confirmed scientifically in this study.

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain.

INTRODUCTION: Two 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABAA/benzodiazepine receptor by comparing them to [(11)C]flumazenil in rhesus monkey. METHODS: Radiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [(18)F]AH114726 and [(18)F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil. RESULTS: [(18)F]GEH120348 and [(18)F]AH114726 were obtained in 13-24% yields (end of synthesis) with high chemical (>95%) and radiochemical (>99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [(18)F]AH114726 and [(18)F]GEH120348 were determined in a non-human primate and directly compared with [(11)C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [(11)C]flumazenil and the known concentrations of GABAA/benzodiazepine receptors in the monkey brain. [(18)F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [(11)C]flumazenil. In contrast, [(18)F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil. CONCLUSION: The ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [(11)C]flumazenil, support that [(18)F]AH114726 is a suitable option for imaging the GABAA receptor.

GABA(A) receptor imaging with positron emission tomography in the human newborn: a unique binding pattern.

Flumazenil is a specific, reversibly bound antagonist at benzodiazepine binding sites of gamma-aminobutyric acid A receptors; these sites can be imaged using positron emission tomography with 11C-flumazenil. We reported an exponential decline of flumazenil volume of distribution (proportional to receptor binding) of gamma-aminobutyric acid A receptors in children 2 to 17 years. Six newborns (33.3-46.7 weeks' postconception) were studied. All had experienced epileptic seizures and undergone 60-minute dynamic 11C-flumazenil-positron emission tomography imaging after injection of 0.4 mCi/kg of 11C-flumazenil. All newborns were scanned during their natural sleep. Binding potential (indicating flumazenil receptor binding) was calculated using Logan-plot analysis. Visual and quantitative analyses showed highest receptor binding in the amygdala-hippocampus region, sensory-motor cortex, thalamus, brainstem and basal ganglia, in that order. Cerebellum and most of the cerebral cortex showed relatively low binding. This is the first demonstration of gamma-aminobutyric acid A receptor binding in human neonates and is strikingly different from that in older children/adults, showing a programmed pattern of expression. The ontogeny data of flumazenil receptor binding from children may contribute to understanding regional differences in synaptic plasticity and improve rational therapeutic use of drugs acting at the gamma-aminobutyric acid A receptor in the pediatric population.

[Flumazenil].

Flumazenil, an imidazobenzodiazepine derivative, has been generally used as an antagonist which antagonizes the hypnotic and sedative effects of benzodiazepines at gamma-amino butyric acid receptors. The anesthetic effects induced by benzodiazepine could be reversed effectively and safely by flumazenil alone, and some have recommended diagnostic usage in intensive care unit and emergency unit as well, for suspected benzodiazepine intoxication. Seizures might follow the use of flumazenil. Benzodiazepine overdose patients who have co-ingested tricyclic and tetracyclic antidepressants are especially at risk for this complication. Therefore, flumazenil could be considered in cases in which quick recovery is required and should be administered intravenously in small, incremental doses.