Chen Wei-Hsi, Chiu Chuang-Hsin, Farn Shiou-Shiow, Cheng Kai-Hung, Huang Yuan-Ruei, Lee Shih-Ying, Fang Yao-Ching, Lin Yu-Hua, Chang Kang-Wei
Isotope Application Division, Institute of Nuclear Energy Research, Taoyuan City 325207, Taiwan.
Department of Nuclear Medicine, Tri-Service General Hospital, Taipei 114202, Taiwan.
Pharmaceuticals (Basel). 2023 May 18;16(5):764. doi: 10.3390/ph16050764.
Studies of the neurobiological causes of anxiety disorders have suggested that the γ-aminobutyric acid (GABA) system increases synaptic concentrations and enhances the affinity of GABA (type A) receptors for benzodiazepine ligands. Flumazenil antagonizes the benzodiazepine-binding site of the GABA/benzodiazepine receptor (BZR) complex in the central nervous system (CNS). The investigation of flumazenil metabolites using liquid chromatography (LC)-tandem mass spectrometry will provide a complete understanding of the in vivo metabolism of flumazenil and accelerate radiopharmaceutical inspection and registration. The main goal of this study was to investigate the use of reversed-phase high performance liquid chromatography (PR-HPLC), coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (ESI-QqQ MS), to identify flumazenil and its metabolites in the hepatic matrix. Carrier-free nucleophilic fluorination with an automatic synthesizer for [F]flumazenil, combined with nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging, was used to predict the biodistribution in normal rats. The study showed that 50% of the flumazenil was biotransformed by the rat liver homogenate in 60 min, whereas one metabolite (M1) was a methyl transesterification product of flumazenil. In the rat liver microsomal system, two metabolites were identified (M2 and M3), as their carboxylic acid and hydroxylated ethyl ester forms between 10 and 120 min, respectively. A total of 10-30 min post-injection of [F]flumazenil showed an immediate decreased in the distribution ratio observed in the plasma. Nevertheless, a higher ratio of the complete [F]flumazenil compound could be used for subsequent animal studies. [F] According to in vivo nanoPET/CT imaging and ex vivo biodistribution assays, flumazenil also showed significant effects on GABA receptor availability in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, indicating the formation of metabolites. We reported the completion of the biotransformation of flumazenil by the hepatic system, as well as [F]flumazenil's potential as an ideal ligand and PET agent for the determination of the GABA/BZR complex for multiplex neurological syndromes at the clinical stage.
焦虑症的神经生物学原因研究表明，γ-氨基丁酸（GABA）系统可增加突触浓度，并增强GABA（A 型）受体对苯二氮䓬配体的亲和力。氟马西尼可拮抗中枢神经系统（CNS）中GABA/苯二氮䓬受体（BZR）复合物的苯二氮䓬结合位点。使用液相色谱（LC）-串联质谱法对氟马西尼代谢物进行研究，将有助于全面了解氟马西尼的体内代谢情况，并加速放射性药物的检测和注册。本研究的主要目的是研究使用反相高效液相色谱（PR-HPLC）结合电喷雾电离三重四极杆串联质谱（ESI-QqQ MS）来鉴定肝基质中的氟马西尼及其代谢物。使用自动合成器对[F]氟马西尼进行无载体亲核氟化，并结合纳米正电子发射断层扫描（NanoPET）/计算机断层扫描（CT）成像，来预测正常大鼠体内的生物分布。研究表明，50%的氟马西尼在60分钟内被大鼠肝脏匀浆生物转化，而一种代谢物（M1）是氟马西尼的甲基酯交换产物。在大鼠肝脏微粒体系统中，分别在10至120分钟之间鉴定出两种代谢物（M2和M3），它们分别为羧酸和羟基化乙酯形式。注射[F]氟马西尼后10至30分钟内，血浆中的分布率立即下降。然而，较高比例的完整[F]氟马西尼化合物可用于后续动物研究。根据体内纳米PET/CT成像和体外生物分布测定，氟马西尼对大鼠大脑杏仁核、前额叶皮质、皮质和海马体中的GABA受体可用性也有显著影响，表明有代谢物形成。我们报告了肝脏系统对氟马西尼生物转化的完成情况，以及[F]氟马西尼作为理想配体和PET剂在临床阶段用于测定多重神经综合征的GABA/BZR复合物的潜力。
