Desenvolvimento do radiofármaco [18F] flumazenil para realização de exames PET/CT

Abstract

Molecular Imaging is a technique that allows visualization, characterization and quantification of biochemical processes in a molecular and cellular level, in humans and other live organisms. Among all the technics available, it s possible to highlight PET (Positron Emission Tomography), which needs the administration of a radiotracer in the organism to be studied. Radiotracers or radiopharmaceuticals are molecules that possess a radioactive element in their composition. Nowadays, the most used PET radiopharmaceutical is fluorodeoxyglucose (18F) or [18F]FDG. This molecule is a glucose analogue that accumulates inside the cell, allowing glucose metabolism visualization. However, considering the use of glucose by the brain as its main energy source, this radiopharmaceutical accumulates in high rates in the normal brain, making difficult to see pathological processes. In order to visualize more specific alterations in the brain, many others radiotracers can be used, for example flumazenil (18F). Flumazenil (FMZ) shows high affinity for the benzodiazepine site in the GABAA receptor, acting as competitive antagonist. It s already known that GABAA receptors play a key role in neuronal excitability control, and expression deficiencies in these receptors are involved in many neurological and psychiatric disorders, such as epilepsy, anxiety, depression, schizophrenia, etc. Regarding epilepsy, it is believed that in the epileptogenic foci the GABAA receptor expression is reduced, the brain region where seizures start. For this reason, the aim of this work was to study flumazenil (18F) synthesis, as well as its purification and quality control analysis, in order to produce a molecule that can be used to localize the epileptogenic foci. The fluoride ion (18F-) was produced using PET Trace 16 MeV cyclotron from GE Healthcare, through the nuclear reaction 18O(p,n)18F, through the irradiation of enriched 18O water with accelerated protons. The flumazenil (18F) synthesis was performed in the automated synthesis module TRACERlab FX F-N from GE Healthcare, by nucleophilic aromatic substitution reaction. The established ideal reaction conditions were 145°C for 15 minutes, and 6,2mg precursor mass. The 18F- incorporation degree in the flumazenil molecule was 72±6% (n = 5), verified by reaction mixture analysis. In the last step flumazenil (18F) was purified through high performance liquid chromatography (HPLC) and reserve-phase tC18 cartridge, obtaining a product with a purity higher than 99%. The radionuclidic purity and identity were analyzed by gamma ray spectroscopy and half-life evaluation. Radiochemical purity was verified using thin layer chromatography (TLC) and HPLC. The chemical purity tested the presence of kryptofix 2.2.2, through a colorimetric test, and residual solvents (ethanol and acetonitrile) through gas chromatography (GC). The pH was verified using strips. All the results complied with the pharmacopeia using [18F]FDG as reference. Synthesis time was 80 minutes including purification steps and the overall yield was 9.3% (decay corrected). The radiopharmaceutical stability was analyzed for 8 hours, and no impurities were detected in this period. The method developed showed to be viable to produce flumazenil (18F), which can be used in pre-clinical and clinical studies in the future. The knowledge acquired with this work will allow the improvement of this technology in the country, and the research of new radiotracers for PET/CT imaging.