Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging.

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 GABAA (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 [18F]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 [18F]flumazenil showed an immediate decreased in the distribution ratio observed in the plasma. Nevertheless, a higher ratio of the complete [18F]flumazenil compound could be used for subsequent animal studies. [18F] According to in vivo nanoPET/CT imaging and ex vivo biodistribution assays, flumazenil also showed significant effects on GABAA 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 [18F]flumazenil's potential as an ideal ligand and PET agent for the determination of the GABAA/BZR complex for multiplex neurological syndromes at the clinical stage.

Automated Synthesis of [18F]Flumazenil Application in GABAA Receptor Neuroimaging Availability for Rat Model of Anxiety.

Clinical studies have demonstrated that the γ-aminobutyric acid type A (GABAA) receptor complex plays a central role in the modulation of anxiety. Conditioned fear and anxiety-like behaviors have many similarities at the neuroanatomical and pharmacological levels. The radioactive GABA/BZR receptor antagonist, fluorine-18-labeled flumazenil, [18F]flumazenil, behaves as a potential PET imaging agent for the evaluation of cortical damage of the brain in stroke, alcoholism, and for Alzheimer disease investigation. The main goal of our study was to investigate a fully automated nucleophilic fluorination system, with solid extraction purification, developed to replace traditional preparation methods, and to detect underlying expressions of contextual fear and characterize the distribution of GABAA receptors in fear-conditioned rats by [18F]flumazenil. A carrier-free nucleophilic fluorination method using an automatic synthesizer with direct labeling of a nitro-flumazenil precursor was implemented. The semi-preparative high-performance liquid chromatography (HPLC) purification method (RCY = 15-20%) was applied to obtain high purity [18F]flumazenil. Nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging and ex vivo autoradiography were used to analyze the fear conditioning of rats trained with 1-10 tone-foot-shock pairings. The anxiety rats had a significantly lower cerebral accumulation (in the amygdala, prefrontal cortex, cortex, and hippocampus) of fear conditioning. Our rat autoradiography results also supported the findings of PET imaging. Key findings were obtained by developing straightforward labeling and purification procedures that can be easily adapted to commercially available modules for the high radiochemical purity of [18F]flumazenil. The use of an automatic synthesizer with semi-preparative HPLC purification would be a suitable reference method for new drug studies of GABAA/BZR receptors in the future.

Imaging and biodistribution of radiolabeled SP90 peptide in BT-483 tumor bearing mice.

The objective of this study was to evaluate radiolabeled DOTA-SP90 as a radiotracer for breast cancer. The in vitro competition assay showed that radiolabeled DOTA-SP90 had significant binding affinity to BT-483 cancer cells. Biodistribution, nanoSPECT/CT and nanoPET/CT imaging results indicated that radiolabeled DOTA-SP90 can accumulate in tumors. In addition, radiolabeled DOTA-SP90 peptides can also detect metastatic tumors. Therefore, radiolabeled SP90 peptide may provide the potential capability as diagnostic agent for breast cancer patients.

Clinical and Electrophysiological Analysis of Chronic Eyelid Twitching.

PURPOSE: Nearly everyone had experience of eyelid twitching (ET) and most physicians think it is benign and self limited. Most neurologists consider it is a symptom rather a disease. However, sometimes ET persists longer and becomes bothersome in some cases, i.e. chronic ET (CET). CET was seldom discussed seriously and studied extensively. Few studies concerning of CET especially its electrophysiological features had been reported. The purposes of this study are to delineate its clinical features and electrophysiological characters of CET. In consequence by knowing its pathophysiology we can prove CET is a disease entity as minor form of facial nerve neuropathy. METHODS: A retrospective study by reviewing medical charts of patients with facial twitching and have been received examinations of facial nerve latency and blink response. We defined CET as persisting of ET more than 2 weeks. We collected 142 patients, age from 12 to 73 years-old, 34 men and 108 women. The onset month and season, sex, abnormal side, and abnormal index (AI=abnormal side minus normal side/normal side) of several variables from electrophysiological study were compared between men and women of CET. RESULTS: There was significant gender difference on CET. Women were more vulnerable than men (female to male: 3 to 1). CET tended to develop more in cold weather (61.27%). Half of CET cases demonstrated delayed or absent R2 response in blink reflex. And 45.8% cases had prolonged facial nerve latency (>5% side to side difference), with the rate higher in women (48.5% vs 38.2%). These two findings indicating conduction defect of facial nerve pathway in CET cases. CONCLUSION: Although CET has been considered as a benign, transient, somewhat physiological phenomenon, our study may suggest it can be a disease entity with minor facial nerve neuropathy.

Correlation of hematologic factors to carotid intima-media thickness in men and women: a study of 2767 asymptomatic subjects of Taiwan.

PURPOSE: A correlation between carotid intima-media thickness (CIMT) and blood cells has not been well documented. Studies of a possible relationship between blood cell components and left (Lt) and right (Rt) side CIMT in asymptomatic men and women in Taiwan have not been conducted previously. The study aims to correlate factors of complete blood cells (CBC) to CIMT for men and women. METHODS: Data collection from 2767 asymptomatic healthy checkup individuals, age 35-75, 1517 men and 1250 women, were separated into four groups: CIMTML (men's Lt), CIMTMR (men's Rt), CIMTWL (women's Lt), and CIMTWR (women's Rt) for analysis of CBC factors vs. CIMT. A cut-off point for each factor was examined for the existence of significant differences in CIMT for individuals among the four test groups who had abnormal CBC data. RESULTS: The regression equations of eight CBC factors vs. CIMT of four groups calculated as R2adj range were from 70.5% to 79.4%. Further comparison of CIMT in different groups by cut points of CBC factors showed significant differences in red blood cells (RBC) and platelets (PLT), but not in white blood cells (WBC). CONCLUSION: Higher RBC, and lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were associated with lower CIMT in asymptomatic men in Taiwan. Further studies to confirm these differences are warranted.

The age correlation of the carotid intima-media thickness according to sex and side in asymptomatic subjects.

PURPOSE: Reports concerning carotid intima-media thickness (CIMT) and linear correlation to age in healthy subjects did not distinguish the side and sex of the subjects. The purpose of this investigation attempts to clarify these issues. METHODS: 2402 asymptomatic persons, age 35-64, are separated into men's left (Lt) and right (Rt) and women's Lt and Rt carotid arteries for difference of CIMT between them and analysis of CIMT vs. age. RESULTS: There are significant difference between men's CIMT of Lt(CIMTML) vs Rt (CIMTMR), women's Lt(CIMTWL) vs. Rt (CIMTWR), Lt side CIMT of men vs women, and Rt side CIMT of men vs. women. The regression equation of CIMT vs. age for all four groups is determinated. CONCLUSION: We found an excellent linear correlation of CIMT to age and CIMT is significantly higher in men than women, so as higher in Lt than Rt. Further grouping of data into about 5-year period showed more clearly stepwise increasing of CIMT, so as the ratios of Lt CIMT different than Rt. CIMT study is served as highly efficient examination in therapy, prevention, clinic, or research survey about atherosclerosis and risk of stroke. Future study design concerning CIMT in separation groups of men and women, so as Lt and Rt is highly recommended.

Development of acute and subacute toxicity with the serotonin transporter radiopharmaceutical, ADAM.

It is predicted that depression will become the most common neurological disease in the new millennium. Its incidence is currently about 3% of diseases worldwide. Serotonin is an essential neurotransmitter for the central and peripheral nervous systems and plays a crucial role in neuropsychiatric disorders. (123)I-labeled ADAM was developed to facilitate an early diagnosis of serotonin transporter (SERT) abnormalities in the brain. Many studies have confirmed that the binding of this radiotracer to SERTs is associated with depression. The aim of this study was to evaluate the acute and subacute toxicity of ADAM and to determine its no observed adverse effect level (NOAEL) by administering it via intravenous injection to Sprague-Dawley rats for 14 consecutive days. None of the animals died, and no treatment-related clinical signs were observed. Urinalysis, hematology, and clinical chemistry analysis revealed that daily administration of ADAM (2-2-dimethylaminomethylphenylthio-5-iodophenylamine) for 2 weeks had no toxicological effects. It is concluded that ADAM exerts no adverse toxic effects on this animal model. The NOAEL was 155 microg/kg/day.

Acute toxicity of two Alzheimer's disease radiopharmaceuticals: FDDNP and IMPY.

Alzheimer's disease (AD) is a neurodegenerative disorder that results in memory deficits. The effect of AD is the leading cause of dementia in the United States and constitutes a burgeoning public health problem. AD is characterized by the presence of two aberrant structures, senile plaques, and neurofibrillary tangles, present in the brain of the patients. [(18)F]FDDNP and [(123)I]IMPY were developed for the early diagnosis of AD by Dr. J. Barrios and Dr. H. Kung, respectively. These two radiotracers could bind with the amyloid location site in the AD patient brain. The aim of this study was to analyze the acute single toxic effects dose of two nonradiochemical labeled compounds in rats. Animals were injected from the tail vein with nonlabeled-FDDNP (0- 5 mg/kg) and nonlabeled-IMPY (0-300 microg/kg), respectively, and observed for 2 weeks. These doses provide safety margins of 35,000- to 140-fold and 1,000- to 100-fold over the maximal recommend human dose (0.1 mg/70 kg) and (20 microg/60 kg) (by FDDNP and IMPY), respectively. With IMPY, there were no changes in mortality, clinical situation, and gross necropsy. With FDDNP, the high dose (5 mg/kg) produced mortality in 2 of 5 and 1 of 5 in male and female rats, respectively. The high dose of FDDNP showed liver damage in dying animals. No other adverse toxic effects at dose levels up to 1.0 mg/kg of FDDNP were noted. FDDNP exerted no adverse toxic effects in rats given doses up to 1 mg/kg and IMPY at the dose levels up to 300 microg/kg.

Enhanced interferon signaling pathway in oral cancer revealed by quantitative proteome analysis of microdissected specimens using 16O/18O labeling and integrated two-dimensional LC-ESI-MALDI tandem MS.

Oral squamous cell carcinoma (OSCC) remains one of the most common cancers worldwide, and the mortality rate of this disease has increased in recent years. No molecular markers are available to assist with the early detection and therapeutic evaluation of OSCC; thus, identification of differentially expressed proteins may assist with the detection of potential disease markers and shed light on the molecular mechanisms of OSCC pathogenesis. We performed a multidimensional (16)O/(18)O proteomics analysis using an integrated ESI-ion trap and MALDI-TOF/TOF MS system and a computational data analysis pipeline to identify proteins that are differentially expressed in microdissected OSCC tumor cells relative to adjacent non-tumor epithelia. We identified 1233 unique proteins in microdissected oral squamous epithelia obtained from three pairs of OSCC specimens with a false discovery rate of <3%. Among these, 977 proteins were quantified between tumor and non-tumor cells. Our data revealed 80 dysregulated proteins (53 up-regulated and 27 down-regulated) when a 2.5-fold change was used as the threshold. Immunohistochemical staining and Western blot analyses were performed to confirm the overexpression of 12 up-regulated proteins in OSCC tissues. When the biological roles of 80 differentially expressed proteins were assessed via MetaCore analysis, the interferon (IFN) signaling pathway emerged as one of the most significantly altered pathways in OSCC. As many as 20% (10 of 53) of the up-regulated proteins belonged to the IFN-stimulated gene (ISG) family, including ubiquitin cross-reactive protein (UCRP)/ISG15. Using head-and-neck cancer tissue microarrays, we determined that UCRP is overexpressed in the majority of cheek and tongue cancers and in several cases of larynx cancer. In addition, we found that IFN-beta stimulates UCRP expression in oral cancer cells and enhances their motility in vitro. Our findings shed new light on OSCC pathogenesis and provide a basis for the future development of novel biomarkers.

Imaging the availability of serotonin transporter in rat brain with 123I-ADAM and small-animal SPECT.

BACKGROUND: Imaging serotonin transporters during antidepressant treatment in small animals is a useful tool for preclinical study during drug development. In this work, we aimed to demonstrate the feasibility of using 123I-ADAM and small-animal SPECT to monitor serotonin transporter availabilities in rat brains prior to and after administration of a selective serotonin re-uptake inhibitor. METHODS: Male Sprague-Dawley rats with and without administration of citalopram (4 mg x kg body weight) were examined in this study. During the process rat brains were scanned using a double-headed microSPECT system equipped with pinhole collimators. SPECT tomographic images and X-ray computed tomography (CT) were acquired after introducing 123I-ADAM via the tail vein. The 123I-ADAM specific binding was assessed by SPECT/CT fused image to draw regions of interest in the midbrain and cerebellum. Ex-vivo autoradiography was carried out as a parallel investigation to validate the SPECT technique. RESULTS: SPECT images displayed specific binding ratio in midbrain to be 0.91+/-0.30 averaged from three rats. Drug occupancies (95.47+/-1.56)% were shown after administration of citalopram in a dosage of 4 mg x kg. CONCLUSION: This study demonstrated that the serotonin transporter availability during antidepressant treatment in small animals can be assessed semi-quantitatively by using 123I-ADAM and SPECT.