Unlabeled Flumazenil Research Articles

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. MethodsThe 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. ResultsNo 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. ConclusionReliable 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.

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

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. 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. [(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. 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.

Simultaneous in vivo measurements of receptor density and affinity using [11C]flumazenil and positron emission tomography: Comparison of full saturation and steady state methods

The binding of PET radiotracer [(11)C]flumazenil to the GABA(A) receptors is described by the receptor density (B(max)) and binding affinity (K(D)). The estimation of B(max) and K(D) is usually based on Scatchard analysis including at least two PET scans at steady state of various specific activities. Recently, a novel full saturation method to estimate both B(max) and K(D) was proposed, in which a saturating dose of flumazenil is given to cover a wide range of different receptor occupancies within a single scan. The aim of the present study was a direct comparison of steady state and full saturation methods for determining B(max) and K(D) of [(11)C]flumazenil in the same group of male Sprague-Dawley rats. Fourteen rats underwent 3 consecutive [(11)C]flumazenil scans of 30 min duration each. A tracer dose was injected at the start of the first scan. Prior to the second scan the tracer was mixed with 5, 20, 100 or 500 μg unlabelled (cold) flumazenil to cover a wide range of receptor occupancies during the scan. The third scan was performed during a constant intravenous infusion of unlabelled flumazenil, resulting in ~50% GABA(A) receptor occupancy. The first and third scans were part of the steady state method, whilst the second scan was performed according to the full saturation method. For both methods, B(max) and K(D) were then derived by compartmental modelling. Both methods yielded similar B(max) and K(D) estimates. The full saturation method yielded B(max) values of 37 ± 5.8 ng · mL(-1) and K(D) values of 7.6 ± 2.0 ng · mL(-1), whilst the steady state method yielded B(max) values of 33 ± 5.4 ng · mL(-1) and K(D) values of 7.1 ± 0.8 ng · mL(-1). The main advantage of the full saturation method is that B(max) and K(D) can be obtained from a single PET scan.

Preparation of highly specific radioactivity [ 18F]flumazenil and its evaluation in cynomolgus monkey by positron emission tomography

A straightforward method for the preparation of no-carrier-added (n.c.a.) [ 18F]flumazenil via standard nucleophilic radiofluorination of the corresponding nitro-analog Ro 15-2344 has been developed. The labeling was performed by employing the K 18F/kryptofix complex in DMF at 160°C for 30 min and equimolar ratio [K/K2.2.2] +18F −/precursor. Under these conditions, an 18F incorporation rate into flumazenil was in the range of 55–60%. The final product was isolated by HPLC purification within a total synthesis time of 75 min and a radiochemical yield of about 30% (EOB). Human post-mortem whole-hemisphere autoradiography of brain sections demonstrated selective uptake of the radioligand in the areas of high density of the central benzodiazepine receptors (BZR). PET studies in a cynomolgus monkey and metabolite studies by HPLC demonstrated similar results by [ 18F]flumazenil as for [ 11C]flumazenil. In blocking experiments, almost all radioactivity was inhibited by the addition of unlabeled flumazenil. [ 18F]Flumazenil is a suitable radioligand for PET assessment of the BZR.

Determination of flumazenil in human plasma by liquid chromatography–electrospray ionisation tandem mass spectrometry

A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method was developed to determine unlabelled flumazenil (Ro 15-1788) in human plasma in [ 11C]flumazenil positron emission tomography (PET) studies. N-Methyl tri-deuterated flumazenil was used as an internal standard. The analyte and internal standard were extracted from plasma samples using solid-phase extraction, with a recovery of 78%. This was determined through the convenience of radioactivity measurements of 11C-labelled flumazenil. The evaporated and reconstituted eluate was analysed by LC–ESI–MS/MS. The calibration curve was linear over the tested concentration range of 0.05–0.5 nM (15–150 pg/ml) with a correlation coefficient, R 2, of 0.998±0.001. A high precision was achieved, with mean intra-assay and inter-assay relative standard deviations of at most 6 and 7%, respectively. The accuracy of the method ranged from 95 to 104%. As a proof of concept, the validated method was applied in the determination of flumazenil in plasma from two healthy volunteers participating in a PET study with three repeated investigations. A bolus-infusion protocol was used to achieve a constant concentration level of flumazenil. The average plasma concentrations ranged from 0.11 and 0.19 nM and all measurements were within the calibration standard range. The flumazenil concentrations were relatively constant within each scan and the average intra-scan precision was 15%.

Evaluation of the Reference Tissue Models for PET and SPECT Benzodiazepine Binding Parameters

Recently, reference tissue methods have been proposed to estimate binding potential from PET data. A reference region without specifically bound ligand is used as an indirect input function to enable the expression of the time–concentration curve of a region of interest using a compartment model. However, PET dopaminergic and serotoninergic studies have shown differences between binding potential (BP) values obtained with reference tissue methods and those obtained with conventional kinetic modeling using an arterial input function. In this study, we measured the BP values for the benzodiazepine receptors in seven subjects using PET [ 11 C]flumazenil and SPECT [ 123 I]iomazenil radioligands. We compared the BP values obtained using the reference tissue methods with those obtained using the conventional kinetic method. These values were also compared with the absolute value of receptor density, B max ′ . For the PET studies, a multi-injection approach employing labeled and unlabeled flumazenil was used to estimate the main binding parameters, BP and B max ′ . For SPECT studies, a single injection protocol of [ 123 I]iomazenil was used to estimate BP values. The BP values were estimated using one- and two-tissue compartment models for the target region. Similar BP values were obtained using either the one- or two-tissue compartment model. This is probably due to the rapid equilibrium between tissue compartments reached with these radioligands. For PET and SPECT, these BP values were highly correlated ( r > 0.960) to the BP values obtained using the arterial input function. We also found high correlations between the BP values obtained using the simplified reference tissue method and the receptor density parameter B max ′ ( r > 0.884). However, the reference tissue methods yielded lower BP values than those obtained using the conventional approach. Moreover, there was a bias on BP values that was not a simple scaling. It seems that the physiological values found in gray matter structures using these radioligands give acceptable BP values. We conclude that the reference tissue methods should be carefully evaluated for each radioligand.

Evaluation of the Reference Tissue Models for PET and SPECT Benzodiazepine Binding Parameters

Recently, reference tissue methods have been proposed to estimate binding potential from PET data. A reference region without specifically bound ligand is used as an indirect input function to enable the expression of the time–concentration curve of a region of interest using a compartment model. However, PET dopaminergic and serotoninergic studies have shown differences between binding potential (BP) values obtained with reference tissue methods and those obtained with conventional kinetic modeling using an arterial input function. In this study, we measured the BP values for the benzodiazepine receptors in seven subjects using PET [ 11C]flumazenil and SPECT [ 123I]iomazenil radioligands. We compared the BP values obtained using the reference tissue methods with those obtained using the conventional kinetic method. These values were also compared with the absolute value of receptor density, B max ′. For the PET studies, a multi-injection approach employing labeled and unlabeled flumazenil was used to estimate the main binding parameters, BP and B max ′. For SPECT studies, a single injection protocol of [ 123I]iomazenil was used to estimate BP values. The BP values were estimated using one- and two-tissue compartment models for the target region. Similar BP values were obtained using either the one- or two-tissue compartment model. This is probably due to the rapid equilibrium between tissue compartments reached with these radioligands. For PET and SPECT, these BP values were highly correlated ( r > 0.960) to the BP values obtained using the arterial input function. We also found high correlations between the BP values obtained using the simplified reference tissue method and the receptor density parameter B max ′ ( r > 0.884). However, the reference tissue methods yielded lower BP values than those obtained using the conventional approach. Moreover, there was a bias on BP values that was not a simple scaling. It seems that the physiological values found in gray matter structures using these radioligands give acceptable BP values. We conclude that the reference tissue methods should be carefully evaluated for each radioligand.

18F]Fluoroethylflumazenil: a novel tracer for PET imaging of human benzodiazepine receptors.

5-(2'-[18F]Fluoroethyl)flumazenil ([18F]FEF) is a fluorine-18 labelled positron emission tomography (PET) tracer for central benzodiazepine receptors. Compared with the established [11C]flumazenil, it has the advantage of the longer half-life of the fluorine-18 label. After optimisation of its synthesis and determination of its in vitro receptor affinities, we performed first PET studies in humans. PET studies in seven healthy human volunteers were performed on a Siemens ECAT EXACT whole-body scanner after injection of 100-280 MBq [L8F]FEF. In two subjects, a second PET scan was conducted after pretreatment with unlabelled flumazenil (1 mg or 2.5 mg i.v., 3 min before tracer injection). A third subject was studied both with [18F]FEF and with [11C]flumazenil. Brain radioactivity was measured for 60-90 min p.i. and analysed with a region of interest-oriented approach and on a voxelwise basis with spectral analysis. Plasma radioactivity was determined from arterial blood samples and metabolites were determined by high-performance liquid chromatography. In human brain, maximum radioactivity accumulation was observed 4 +/- 2 min p.i., with a fast clearance kinetics resulting in 50% and 20% of maximal activities at about 10 and 30 min, respectively. [18F]FEF uptake followed the known central benzodiazepine receptor distribution in the human brain (occipital cortex >temporal cortex >cerebellum >thalamus >pons). Pretreatment with unlabelled flumazenil resulted in reduced tracer uptake in all brain areas except for receptor-free reference regions like the pons. Parametric images of distribution volume and binding potential generated on a voxelwise basis revealed two- to three-fold lower in vivo receptor binding of [18F]FEF compared with [11C]flumazenil, while relative uptake of [18F]FEF was higher in the cerebellum, most likely owing to its relatively higher affinity for benzodiazepine receptors containing the alpha6 subunit. Metabolism of [18F]FEF was very rapid. Polar metabolites represented about 50%-60% of total plasma radioactivity at 5 min and 80%-90% at 20 min p.i. Although [11C]flumazenil has some advantages over [18F]FEF (higher affinity, slower metabolism, slower kinetics), our results indicate that [18F]FEF is a suitable PET ligand for quantitative assessment of central benzodiazepine receptors, which can be used independently of an on-site cyclotron.

Binding of 5-(2′-[ 18F]fluoroenthyl)flumazenil to central benzodiazepine receptors measured in living baboon by positron emission tomography

5-(2′-[ 18F]Fluoroethyl)flumazenil ([ 18F]FEF), a fluorine-18-labeled analogue of the benzodiazepine antagonist flumazenil, was evaluated for use with positron emission tomography (PET). PET imaging of a baboon after i.v. injection of [ 18F]FEF showed that the radiofluorinated ligand rapidly localized in vivo within benzodiazepine receptor-rich cerebral tissues, and that selective disposition was retained for over 2 h. Coinjection of unlabeled flumazenil (0.55 mg/kg i.v.) abolished the heterogeneous cerebral distribution of the tracer; receptor-specific uptake was reduced by approximately 95%. The fluorinated benzodiazepine antagonist was degraded in vivo only to polar radiometabolites that do not cross the blood-brain barrier. [ 18F]FEF has advantages over existing PET radiopharmaceuticals, and is a promising radioligand for non-invasive evaluation of central benzodiazepine receptor binding in vivo.

Plasma pharmacokinetics and metabolism of the benzodiazepine antagonist [11C] Ro 15-1788 (flumazenil) in baboon and human during positron emission tomography studies.

Flumazenil is a specific antagonist of the central benzodiazepine receptor (CBZR). Labelled with 11C, this compound is the reference radioligand for positron emission tomography (PET) study of the CBZR in humans and primates. The time-course of [11C]-flumazenil radioactivity and its main acid metabolite [11C] Ro 15-3890 were reconstructed from the time-course of total radioactivity in plasma after administration with high or low SRA in primates and humans, applying an extraction procedure validated by TLC. The measured pharmacokinetics of [11C]-flumazenil (T1/2 beta = 45.1 +/- 12.3 min, T1/2 alpha = 1.5 +/- 1.5 min; K = 0.14 +/- 0.14 min-1; Vd area = 44.0 +/- 17.0 l; Clp = 40.0 +/- 8.5 l/h) exhibited a very rapid distribution phase followed by fast elimination, with a large volume of distribution; these results were confirmed by HPLC determinations and agree with previously published data on unlabelled flumazenil. Pharmacokinetics of [11C] Ro 15-3890 acid metabolite show that high drug concentrations in the blood are promptly achieved (kf = 0.13 +/- 0.004 min-1), with a very rapid elimination half-life (T1/2m = 4.47 +/- 1.31 min) comparable to that of [11C]-flumazenil. The percentage metabolization of parent compound to the acid [11C] Ro 15-3890 was constant from the 15th minute and was significantly higher in man compared to the monkey. This percentage was increased by prior eating. The other putative metabolites, i.e. labelled [11C] Ro 15-4965 and unlabelled Ro 15-5528, were never observed at detectable concentrations with TLC and HPLC in rabbit, baboon and human blood samples. This pharmacokinetic study of plasma flumazenil may be useful to implement a dynamic method of CBZR quantification using PET and for analysis of pharmacokinetics in brain tissue.

Saturation analysis of specific 11C Ro 15‐1788 binding to the human neocortex using positron emission tomography

AbstractThe 11C‐labelled benzodiazepine antagonist Ro 15–1788 (flumazenil) and positron emission tomography (PET) were used to determine quantitative characteristics of benzodiazepine receptor binding in the neocortex of healthy young men. Saturating doses of unlabelled flumazenil administered i.v., before or together with the ligand‐reduced 11C‐flumazenil accumulation in the neocortex by about 90 per cent. Saturating doses of unlabelled flumazenil had little effect on the accumulation of radioactivity in the benzodiazepine receptor‐poor regions such as pons or white matter. By giving graded doses of unlabelled flumazenil together with the tracer, saturation isotherms were obtained allowing the calculation of receptor density (Bmax) and equilibrium dissociation constant (Kd) values on the basis of certain assumptions Bmax values were in the order of 90 pmol/g and Kd values in the order of 10 nM in the neocortex. Scatchard and Hill plots of the radioactivity data indicated that 11C‐flumazenil binds to saturable sites of a homogeneous population. The data indicate that intravenous doses of 1 or 2 mg flumazenil result in a benzodiazepine receptor occupancy of about 50 per cent. The method described should be useful for studying regional differences in benzodiazepine receptor characteristics in the living human brain in healthy subjects and neuropsychiatric disorders, and also in relation to treatment with drugs interacting with benzodiazepine receptors.