Comparison [(3)H]-flumazenil binding parameters in rat cortical membrane using different separation methods, filtration and centrifugation.

Radioligand receptor binding assays are a common method to evaluate the affinity of newly synthesized benzodiazepine ligands for the receptor. [(3)H]-flumazenil is an antagonist of benzodiazepine receptors and is generally used as a radioligand. In this study, the binding parameters of [(3)H]-flumazenil to rat cortical membranes were evaluated using two separation methods: filtration with GF/C filters and centrifugation. Additionally, the effects of vacuum pressure, exposure time to the cocktail, and geometry on the filtration method were studied. The binding parameters of [(3)H]-flumazenil (Kd and Bmax) were determined through saturation studies using two methods. The results from this study showed that the filtration method is time consuming and requires more steps to be completed. Because filtration causes partial elution of bound [(3)H]-flumazenil into the liquid scintillation cocktail, the results are not reproducible, which result in inaccurate estimation of the binding parameters. The centrifugation method in contrast to filtration is straightforward and produces reproducible as well as reliable results, all of the steps are performed in a single polypropylene tube, which eliminates the loss of tissue and avoids other systematic errors associated with transfer and handling.

Improved work-up procedure for the production of [(18)F]flumazenil and first results of its use with a high-resolution research tomograph in human stroke.

INTRODUCTION: The central benzodiazepine receptor (cBZR)-gamma-aminobutyric acid (GABA(A)) receptor complex in the human brain plays an important role in many neurological and psychiatric disorders. (18)F-Labeled flumazenil ([(18)F]FZ) provides a potentially useful tracer to investigate those disorders by means of positron emission tomography (PET). METHODS: [(18)F]Flumazenil was synthesized from its nitro-precursor Ro 15-2344 in DMF at high temperatures between 150 degrees C and 160 degrees C. Other solvents like acetonitrile and dimethylsulfoxide were also investigated as reaction media. A new HPLC method for the final purification of [(18)F]FZ was developed to circumvent some difficulties associated with a previously published procedure sometimes led to a contamination of [(18)F]FZ with Ro 15-2344. The final purification of the radiotracer was achieved using a Waters Symmetry Prep C18 HPLC column with elution with 0.05 M sodium acetate (NaOAc) buffer (pH 5)/THF/MeOH (80:10:10). RESULTS: [(18)F]FZ could be synthesized in reproducible radiochemical yields (RCYs) of 15-20% (decay corrected to EOB) after 80 min overall synthesis time. The synthesized [(18)F]FZ was applied for the first time in a human PET study in a patient with ischemic right middle cerebral artery stroke using the HRRT high-resolution research scanner (Siemens Medical Solution, Knoxville, TN, USA). CONCLUSIONS: [(18)F]FZ is a potentially useful GABA receptor-binding PET ligand. A modified procedure for its preparation in reproducibly high radiochemical yields has been described and the [(18)F]FZ thus produced has been used successfully in a pilot clinical study.

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry.

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate-methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [18F]fluoroethylflumazenil in routine clinical analysis.

A comparison of methods for the separation of [11C]Ro 15-1788 (flumazenil) from its metabolites in the blood of rabbits, baboons and humans.

The determination of the percentage of unchanged [11C]Ro 15-1788 with respect to total radioactivity, essential for the pharmacokinetic exploitation of data of blood radioactivity decrease obtained in PET studies, may be carried out with equal results using either an extraction procedure or a TLC method. The analysis of blood samples of various sources (rabbits, baboons, healthy volunteers and anxious patients) and the use of an automatic linear scanner to quantify the radioactive distribution on the TLC plates lead to the conclusion that only [11C]Ro 15-1788 and its acid metabolite [11C]Ro 15-3890 and no other radioactive compound are present in blood at detectable concentration.