Reduced SV2A and GABAA receptor levels in the brains of type 2 diabetic rats revealed by [18F]SDM-8 and [18F]flumazenil PET.

PURPOSE: Type 2 diabetes mellitus (T2DM) is associated with a greater risk of Alzheimer's disease. Synaptic impairment and protein aggregates have been reported in the brains of T2DM models. Here, we assessed whether neurodegenerative changes in synaptic vesicle 2 A (SV2A), γ-aminobutyric acid type A (GABAA) receptor, amyloid-ß, tau and receptor for advanced glycosylation end product (RAGE) can be detected in vivo in T2DM rats. METHODS: Positron emission tomography (PET) using [18F]SDM-8 (SV2A), [18F]flumazenil (GABAA receptor), [18F]florbetapir (amyloid-ß), [18F]PM-PBB3 (tau), and [18F]FPS-ZM1 (RAGE) was carried out in 12-month-old diabetic Zucker diabetic fatty (ZDF) and SpragueDawley (SD) rats. Immunofluorescence staining, Thioflavin S staining, proteomic profiling and pathway analysis were performed on the brain tissues of ZDF and SD rats. RESULTS: Reduced cortical [18F]SDM-8 uptake and cortical and hippocampal [18F]flumazenil uptake were observed in 12-month-old ZDF rats compared to SD rats. The regional uptake of [18F]florbetapir and [18F]PM-PBB3 was comparable in the brains of 12-month-old ZDF and SD rats. Immunofluorescence staining revealed Thioflavin S-negative, phospho-tau-positive inclusions in the cortex and hypothalamus in the brains of ZDF rats and the absence of amyloid-beta deposits. The level of GABAA receptors was lower in the cortex of ZDF rats than SD rats. Proteomic analysis further demonstrated that, compared with SD rats, synaptic-related proteins and pathways were downregulated in the hippocampus of ZDF rats. CONCLUSION: These findings provide in vivo evidence for regional reductions in SV2A and GABAA receptor levels in the brains of aged T2DM ZDF rats.

Neuronal Alterations in Secondary Thalamic Degeneration Due to Cerebral Infarction: A 11C-Flumazenil Positron Emission Tomography Study.

BACKGROUND: Studies using animal experiments have shown secondary neuronal degeneration in the thalamus after cerebral infarction. Neuroimaging studies in humans have revealed changes in imaging parameters in the thalamus, remote to the infarction. However, few studies have directly demonstrated neuronal changes in the thalamus in vivo. The purpose of this study was to determine whether secondary thalamic neuronal damage may manifest as a decrease in central benzodiazepine receptors in patients with cerebral infarction and internal carotid artery or middle cerebral artery disease. METHODS: We retrospectively analyzed the data of 140 patients with unilateral cerebral infarction ipsilateral to internal carotid artery or middle cerebral artery disease. All patients had quantitative measurements of 11C-flumazenil binding potential (FMZ-BP), cerebral blood flow, and cerebral metabolic rate of oxygen using positron emission tomography in the chronic stage. Region of interest analysis was performed using NeuroFlexer-an automated region of interest analysis software using NEUROSTAT. RESULTS: In the thalamus ipsilateral to the infarcts, the values of FMZ-BP, cerebral blood flow, and cerebral metabolic rate of oxygen were significantly lower than those in the contralateral thalamus. Significant correlations were found between the ipsilateral-to-contralateral ratio of FMZ-BP and the ipsilateral-to-contralateral ratio of cerebral blood flow or cerebral metabolic rate of oxygen in the thalamus. Patients with corona radiata infarcts and striatocapsular infarcts had significantly decreased ipsilateral-to-contralateral FMZ-BP ratio in the thalamus compared with those without. The ipsilateral-to-contralateral ratio of FMZ-BP in the thalamus was significantly correlated with the ipsilateral-to-contralateral cerebral metabolic rate of oxygen ratio in the frontal cortex and showed a significant negative correlation with the number of perseverative errors on the Wisconsin Card Sorting Test. CONCLUSIONS: Secondary thalamic neuronal damage may manifest as a decrease in central benzodiazepine receptors in patients with cerebral infarction and internal carotid artery or middle cerebral artery disease, which may be associated with frontal lobe dysfunction.

Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAAR is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAARs, affording a seemingly logical site of action, the mechanism(s) by which GABAAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction in vivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAAR-dependent, as it is not inhibited by the GABAAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3Rs (IP3 receptors).

Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

Cellular and molecular signatures of in vivo imaging measures of GABAergic neurotransmission in the human brain.

Diverse GABAergic interneuron networks orchestrate information processing in the brain. Understanding the principles underlying the organisation of this system in the human brain, and whether these principles are reflected by available non-invasive in vivo neuroimaging methods, is crucial for the study of GABAergic neurotransmission. Here, we use human gene expression data and state-of-the-art imaging transcriptomics to uncover co-expression patterns between genes encoding GABAA receptor subunits and inhibitory interneuron subtype-specific markers, and their association with binding patterns of the gold-standard GABA PET radiotracers [11C]Ro15-4513 and [11C]flumazenil. We found that the inhibitory interneuron marker somatostatin covaries with GABAA receptor-subunit genes GABRA5 and GABRA2, and that their distribution followed [11C]Ro15-4513 binding. In contrast, the inhibitory interneuron marker parvalbumin covaried with GABAA receptor-subunit genes GABRA1, GABRB2 and GABRG2, and their distribution tracked [11C]flumazenil binding. Our findings indicate that existing PET radiotracers may provide complementary information about key components of the GABAergic system.

How does α1Histidine102 affect the binding of modulators to α1ß2γ2 GABAA receptors? molecular insights from in silico experiments.

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1ß2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

Region-specific and dose-specific effects of chronic haloperidol exposure on [3H]-flumazenil and [3H]-Ro15-4513 GABAA receptor binding sites in the rat brain.

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABAA receptor (GABAAR) α subunits, including α5GABAAR. Positron emission tomography (PET) measures of GABAAR availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABAAR agonist [3H]-muscimol provide evidence that antipsychotic drugs influence GABAAR availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABAAR radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABAAR subunit-selective radioligand [3H]-Ro15-4513 and the non-subunit selective radioligand [3H]-flumazenil. Chronic haloperidol exposure increased [3H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d=+1.3), which was not dose-dependent. [3H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABAAR radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABAAR-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABAAR in the context of schizophrenia.

Anti-Epileptic Effects of FABP3 Ligand MF1 through the Benzodiazepine Recognition Site of the GABAA Receptor.

Recently, we developed the fatty acid-binding protein 3 (FABP3) ligand MF1 (4-(2-(1-(2-chlorophenyl)-5-phenyl-1H-pyrazol-3-yl)phenoxy) butanoic acid) as a therapeutic candidate for α-synucleinopathies. MF1 shows affinity towards γ-aminobutyric acid type-A (GABAA) receptor, but its effect on the receptor remains unclear. Here, we investigate the pharmacological properties of MF1 on the GABAA receptor overexpressed in Neuro2A cells. While MF1 (1-100 µm) alone failed to evoke GABA currents, MF1 (1 µm) promoted GABA currents during GABA exposure (1 and 10 µm). MF1-promoted GABA currents were blocked by flumazenil (10 µm) treatment, suggesting that MF1 enhances receptor function via the benzodiazepine recognition site. Acute and chronic administration of MF1 (0.1, 0.3 and 1.0 mg/kg, p.o.) significantly attenuated status epilepticus (SE) and the mortality rate in pilocarpine (PILO: 300 mg/kg, i.p.)-treated mice, similar to diazepam (DZP: 5.0 mg/kg, i.p.). The anti-epileptic effects of DZP (5.0 mg/kg, i.p.) and MF1 (0.3 mg/kg, p.o.) were completely abolished by flumazenil (25 mg/kg, i.p.) treatment. Pentylenetetrazol (PTZ: 90 mg/kg, i.p.)-induced seizures in mice were suppressed by DZP (5.0 mg/kg, i.p.), but not MF1. Collectively, this suggests that MF1 is a mild enhancer of the GABAA receptor and exercises anti-epileptic effects through the receptor's benzodiazepine recognition site in PILO-induced SE models.

GABAa receptor density alterations revealed in a mouse model of early moderate prenatal ethanol exposure using [18F]AH114726.

INTRODUCTION: Prenatal ethanol exposure (PEE) has been shown to alter the level and function of receptors in the brain, one of which is GABAa receptors (GABAaR), the major inhibitory ligand gated ion channels that mediate neuronal inhibition. High dose PEE in animals resulted in the upregulation of GABAaR, but the effects of low and moderate dose PEE at early gestation have not been investigated. This study aimed at examining GABAaR density in the adult mouse brain following PEE during a period equivalent to the first 3 to 4 weeks in human gestation. It was hypothesized that early moderate PEE would cause alterations in brain GABAaR levels in the adult offspring. METHODS: C57BL/6J mice were given 10% v/v ethanol during the first 8 gestational days. Male offspring were studied using in-vivo Positron Emission Tomography (PET)/Magnetic Resonance Imaging (MRI), biodistribution, in-vitro autoradiography using [18F]AH114726, a novel flumazenil analogue with a high affinity for the benzodiazepine-binding site, and validated using immunohistochemistry. RESULTS: In vivo PET and biodistribution did not detect alteration in brain tracer uptake. In vitro radiotracer studies detected significantly reduced GABAaR in the olfactory bulbs. Immunohistochemistry detected reduced GABAaR in the cerebral cortex, cerebellum and hippocampus, while Nissl staining showed that cell density was significantly higher in the striatum following PEE. CONCLUSION: Early moderate PEE may induce long-term alterations in the GABAaR system that persisted into adulthood.

GABAA Receptors in the Mongolian Gerbil: a PET Study Using [18F]Flumazenil to Determine Receptor Binding in Young and Old Animals.

PURPOSE: Plastic changes in the central auditory system involving the GABAergic system accompany age-related hearing loss. Such processes can be investigated with positron emission tomography (PET) imaging using [18F]flumazenil ([18F]FMZ). Here, [18F]FMZ PET-based modeling approaches allow a simple and reliable quantification of GABAA receptor binding capacity revealing regional differences and age-related changes. PROCEDURES: Sixty-minute list-mode PET acquisitions were performed in 9 young (range 5-6 months) and 11 old (range 39-42 months) gerbils, starting simultaneously with the injection of [18F]FMZ via femoral vein. Non-displaceable binding potentials (BPnd) with pons as reference region were calculated for auditory cortex (AC), inferior colliculus (IC), medial geniculate body (MGB), somatosensory cortex (SC), and cerebellum (CB) using (i) a two-tissue compartment model (2TCM), (ii) the Logan plot with image-derived blood-input (Logan (BI)), (iii) a simplified reference tissue model (SRTM), and (iv) the Logan reference model (Logan (RT)). Statistical parametric mapping analysis (SPM) comparing young and old gerbils was performed using 3D parametric images for BPnd based on SRTM. Results were verified with in vitro autoradiography from five additional young gerbils. Model assessment included the Akaike information criterion (AIC). Hearing was evaluated using auditory brainstem responses. RESULTS: BPnd differed significantly between models (p < 0.0005), showing the smallest mean difference between 2TCM as reference and SRTM as simplified procedure. SRTM revealed the lowest AIC values. Both volume of distribution (r2 = 0.8793, p = 0.018) and BPnd (r2 = 0.8216, p = 0.034) correlated with in vitro autoradiography data. A significant age-related decrease of receptor binding was observed in auditory (AC, IC, MGB) and other brain regions (SC and CB) (p < 0.0001, unpaired t test) being confirmed by SPM using pons as reference (p < 0.0001, uncorrected). CONCLUSION: Imaging of GABAA receptor binding capacity in gerbils using [18F]FMZ PET revealed SRTM as a simple and robust quantification method of GABAA receptors. Comparison of BPnd in young and old gerbils demonstrated an age-related decrease of GABAA receptor binding.

Quantitative Analysis of [18F]FFMZ and [18F]FDG PET Studies in the Localization of Seizure Onset Zone in Drug-Resistant Temporal Lobe Epilepsy.

BACKGROUND: Positron emission tomography (PET) imaging in epilepsy is an in vivo technique that allows the localization of a possible seizure onset zone (SOZ) during the interictal period. Stereo-electro-encephalography (SEEG) is the gold standard to define the SOZ. The objective of this research was to evaluate the accuracy of PET imaging in localizing the site of SOZ compared with SEEG. METHODS: Seven patients with refractory temporal lobe epilepsy (Ep) and 2 healthy controls (HC) underwent 2 PET scans, one with 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) and another with 2'-[18F]fluoroflumazenil (FFMZ), acquired 1 day apart. FDG was acquired for 10 min (static scan) 1 h after administration. An FFMZ scan was acquired for 60 min from radiopharmaceutical administration in a dynamic mode. Each brain PET image was segmented using a standard template implemented in PMOD 3.8. The pons was used as the reference region for modeling of the nondisplaceable binding potential (BPND)for FFMZ, and to obtain uptake ratios for FDG. SEEG studies of patients were performed as a part of their surgical evaluation to define the SOZ. RESULTS: Well-defined differences between HC and Ep were found with both radiopharmaceuticals, showing the utility to identify abnormal brain regions using quantitative PET imaging. Lateralization of the SOZ findings by PET (lower uptake/binding in a specific brain hemisphere) matched in 86% for FFMZ and 71% for FDG with SEEG data. CONCLUSION: Quantitative PET imaging is an excellent complementary tool that matches reasonably well with SEEG to define SOZ in presurgical evaluation.

Temporal Changes on 123I-Iomazenil and Cerebral Blood Flow Single-photon Emission Computed Tomography in a Patient with Anti-N-methyl-D-aspartate Receptor Encephalitis.

A 45-year-old man was admitted due to tonic seizures, aphasia, disturbance of consciousness, and abnormal behavior. Because cerebral magnetic resonance imaging findings were normal and mild cerebrospinal fluid (CSF) pleocytosis was observed, autoimmune encephalitis was suspected. The presence of anti-N-methyl-D-aspartate (NMDA) receptor antibodies in the CSF was subsequently confirmed. 123I-Iomazenil and cerebral blood flow single photon emission computed tomography (SPECT) revealed an abnormal uptake in the left frontotemporal region. Multimodal immunotherapy was administered, which remarkably improved the level of consciousness. Progressive reversibility of SPECT findings with clinical improvement suggested that the disorder-related functional deficits had been caused by anti-NMDA receptor antibodies.

Relationship between intelligence quotient (IQ) and cerebral metabolic rate of oxygen in patients with neurobehavioural disability after traumatic brain injury.

OBJECTIVE: Cerebral metabolism may play a significant role in neurobehavioural disability following traumatic brain injury (TBI). In this study, we examined the relationship between intelligence quotient (IQ) and the cerebral metabolic rate of oxygen (CMRO2) in the lateral prefrontal cortex, which was measured by 15O-labelled gas positron emission tomography (PET), in patients with TBI. MATERIALS AND METHODS: The subjects were 12 patients (eight males and four females) who suffered from neurobehavioural disability following TBI. Their mean age was 33.3 years. The cause of injury was traffic accidents in all patients and the mean period after injury was 44.8 months. These patients underwent 15O-labelled gas PET and tests using either the Wechsler Adult Intelligence Scale-Revised (WAIS-R) or the Wechsler Intelligence Scale for Children-Revised (WISC-R). Pearson's correlation between CMRO2 and total IQ (TIQ) was calculated. RESULTS: A statistically significant correlation was observed between TIQ and CMRO2 in the right Brodmann areas (BAs) 44 and 45. The lower the WAIS score, the higher the CMRO2 in both areas. CONCLUSION: Neurological function negatively correlated with the metabolism of oxygen. It was possible that changes in brain networks increased the neuronal activity in the undamaged areas and that the increased activity compensated for the function decline.

Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidances from Regulatory Agencies: Focus on CYP3A4 mRNA In Vitro Response Thresholds, Variability, and Clinical Relevance.

The Innovation and Quality Induction Working Group presents an assessment of best practice for data interpretation of in vitro induction, specifically, response thresholds, variability, application of controls, and translation to clinical risk assessment with focus on CYP3A4 mRNA. Single concentration control data and Emax/EC50 data for prototypical CYP3A4 inducers were compiled from many human hepatocyte donors in different laboratories. Clinical CYP3A induction and in vitro data were gathered for 51 compounds, 16 of which were proprietary. A large degree of variability was observed in both the clinical and in vitro induction responses; however, analysis confirmed in vitro data are able to predict clinical induction risk. Following extensive examination of this large data set, the following recommendations are proposed. a) Cytochrome P450 induction should continue to be evaluated in three separate human donors in vitro. b) In light of empirically divergent responses in rifampicin control and most test inducers, normalization of data to percent positive control appears to be of limited benefit. c) With concentration dependence, 2-fold induction is an acceptable threshold for positive identification of in vitro CYP3A4 mRNA induction. d) To reduce the risk of false positives, in the absence of a concentration-dependent response, induction ≥ 2-fold should be observed in more than one donor to classify a compound as an in vitro inducer. e) If qualifying a compound as negative for CYP3A4 mRNA induction, the magnitude of maximal rifampicin response in that donor should be ≥ 10-fold. f) Inclusion of a negative control adds no value beyond that of the vehicle control.

Structure of a human synaptic GABAA receptor.

Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABAA receptor). Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABAA receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1ß2γ2 GABAA receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.

123I-iomazenil whole-body imaging to detect hepatic carboxylesterase drug-metabolizing enzyme activity.

OBJECTIVES: Drugs are mainly metabolized by hepatic enzymes, the activity of which can differ between individuals. Although it is ideal to measure the hepatic clearance of liver-targeted drugs in individualized medicine, blood enzyme tests typically measure metabolic drug clearance in the entire body, and not just in the liver. We investigated whether I-iomazenil imaging can directly assess and quantify the activity of hepatic drug-metabolizing enzymes. MATERIALS AND METHODS: Hepatic enzymes that metabolize I-iomazenil were identified by thin-layer chromatography in mouse liver homogenates with bis(4-nitrophenyl) phosphate (BNPP) inhibitor for carboxylesterase enzymes and nicotinamide adenine dinucleotide phosphate (NADPH) generator for cytochrome P450 enzymes. Whole-body images of mice were acquired using I-iomazenil with and without BNPP, and the distribution was also obtained. The metabolism of I-iomazenil in the blood, liver, gall bladder, and bladder was investigated by thin-layer chromatography. RESULTS: From the in-vitro metabolism of I-iomazenil using BNPP, the enzyme converting I-iomazenil to I-R-COOH was identified as carboxylesterase, and that converting I-iomazenil to M2 was identified as cytochrome P450 in experiments with and without an NADPH generator. The biological distribution and whole-body imaging showed increased accumulation in the liver of mice administered BNPP compared with normal mice, but decreased levels in the gall bladder and small intestine. The main fraction in bile and urine was I-R-COOH, with two unknown metabolites (M1 and M2), I, and I-iomazenil also being present. CONCLUSION: I-iomazenil whole-body imaging has good possibility of direct measurement of hepatic carboxylesterase activity as accumulation of I-R-COOH in the gall bladder through bile and in the bladder through urine.

Increase in extraction of I-123 iomazenil in patients with chronic cerebral ischemia.

BACKGROUND: Cerebral extraction of diffusively distributed substances like oxygen has been suggested to change according to the cerebral blood flow (CBF) and status of the microvasculature. The relationships between the cerebral extraction of diffusively distributed lipophilic tracers and the severity of cerebral ischemia has not yet been clarified. In the present study, we attempted to elucidate the association between the extraction fraction of the lipophilic tracer I-123 iomazenil (IMZ) (IMZ-EF) and the oxygen extraction fraction (OEF) derived from O-15 PET in patients with chronic steno-occlusive disease of internal carotid artery (ICA) or middle cerebral artery (MCA). METHODS: Seven patients with unilateral chronic severe stenosis or occlusion of the middle cerebral/internal cerebral artery were prospectively recruited for this study. All the patients underwent both O-15 PET and quantitative I-123 IMZ SPECT. Parametric images derived from the PET and SPECT scans were anatomically normalized and evaluated by automated image analysis based on the volume-of-interest template. RESULTS: The asymmetry index (AI) of IMZ-EF was shown to be significantly correlated with the AI of OEF (r = 0.562, P < 0.001) in the internal carotid artery perfusion area. Strong and significant correlation between the AI of the influx rate constant K1 of IMZ and the AI of the cerebral metabolic rate of oxygen (r = 0.552, P = 0.001) was clarified. CONCLUSIONS: Our results suggested that the transportation efficiency of I-123 IMZ into the brain tissue was an indicator for evaluating severity of cerebral ischemia in patients with chronic steno-occlusive disease of ICA or MCA. Cerebral metabolic state can possibly be estimated by I-123 IMZ SPECT without cyclotron.

PET Imaging of 18F-FDG, 11C-methionine, 11C-flumazenil, and 11C-4DST in Progressive Multifocal Leukoencephalopathy.

The use of positron emission tomography (PET) imaging in progressive multifocal leukoencephalopathy (PML) has rarely been reported. We herein report a set of PET images in a 63-year-old patient with PML. In PML lesions, the uptake of 18F-fluorodeoxyglucose, 11C-methionine, 11C-flumazenil, and [methyl-11C]4'-thiothymidine was decreased, increased, decreased, and unchanged, respectively. These results suggest that glucose metabolism decreased, protein synthesis increased, neuronal integrity decreased, and the DNA synthesis and cellular proliferation of host cells were not activated in PML lesions. These results may reflect very little infiltration by inflammatory cells and active infection with JC virus in this case.

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.

Abnormal distribution of GABAA receptors in brain of duchenne muscular dystrophy patients.

INTRODUCTION: In this study we sought to: (1) determine the distribution of GABAA receptors (GABAA -Rs) in the brain of Duchenne muscular dystrophy (DMD) patients; and (2) ascertain if the distribution pattern correlates with cognitive dysfunction. METHODS: Fourteen DMD patients [young adult (n = 7, 18-25 years old) and older adult (n = 7, 30-37 years old) groups] and 16 age-matched normal volunteers participated. GABAA -R distribution was assessed using 123 I-IMZ-SPECT. Neuropsychological assessments were performed using 3 different test batteries, the WAIS-III, WMS-R, and Wisconsin Card Sorting Test (WCST). RESULTS: All DMD patients showed significant decline in 123 I-IMZ uptake in the prefrontal cortex (P < 0.05). Although no differences were detected in the WAIS-III and WMS-R, the WCST scores of DMD patients (2.8 ± 1.9) were significantly lower (P < 0.01) than those of normal volunteers (5.4 ± 0.7). Both abnormalities were more pronounced in older adult patients. CONCLUSION: The findings demonstrate that DMD is accompanied by a reduction in the prefrontal cortex distribution of GABAA -Rs. Muscle Nerve 55: 591-595, 2017.