Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging
Abstract
:1. Introduction
2. Results
2.1. High Performance Liquid Chromatography Analysis of Flumazenil
2.2. Mass Spectrometric Analysis of Flumazenil
2.3. Study of Flumazenil Biotransformation by Hepatic Enzyme Systems
2.3.1. Flumazenil Metabolism Study in Rat Liver Homogenate
2.3.2. Flumazenil Metabolism Study in Human Liver Microsomes
2.4. A Summary of the Metabolic Pathways of Flumazenil
2.5. In Vitro Serum Stability of [18F]flumazenil
2.6. In Vivo NanoPET/CT Assay of [18F]flumazenil
2.7. Ex Vivo Biodistribution Assay with [18F]flumazenil
3. Discussion
4. Materials and Methods
4.1. Materials and Reagents
4.2. Animals
4.3. Apparatus and Equipment
4.3.1. High Performance Liquid Chromatography Instrumentation
4.3.2. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry Instrumentation
4.4. Procedures for Metabolism Study
4.4.1. Biotransformation and Pretreatment for High Performance Liquid Chromatography of Flumazenil in Rat Liver Homogenate
4.4.2. Flumazenil Metabolization in Rat Liver Microsomes
4.5. Synthesis of [18F]flumazenil
4.6. In Vitro Serum Stability
4.7. In Vivo NanoPET/CT Neuroimaging Studies
4.8. Ex Vivo Bio-Distribution Assay
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Parameters | |
---|---|
HPLC | |
Stationary phase | ZORBAX Eclipse XDB-C18 4.6 ID × 100 mm, 5 μm thermostat at 25 °C |
Mobile phase | |
Flow rate | 0.6 mL min−1 |
Composition | A: ammonium acetate 5 mM aqueous pH 7.0 with 1% CH3CN |
B: only CH3CN | |
Gradient program | 0–3.5 min, 10% → 40% B |
3.5–10.0 min, 40% B isocratic | |
10.0–10.1 min, 40% → 10% B | |
10.1–15.0 min, 10% B isocratic | |
Detector | DAD at 250 nm |
Turnaround time | 17 min |
Mass Spectrometry | |
Source temperature (°C) | 350 |
Polarity | Positive |
Resolution, Q1 and Q3 | Unit |
Nebulizer gas, NEB (psi) | 40 |
Curtain gas, CUR (psi) | 10 |
Turbo gas | 15 |
Collision gas, CAD (psi) | Medium |
Ion spray voltage, IS (V) | 5000 |
Ion energy 1, IE1 (V) | 0.4 |
Ion energy 3, IE3 (V) | 0.3 |
Declustering potential, DP (V) | 70 |
Entrance potential, EP (V) | 10 |
Detector parameter | Positive |
-Channel electron multiplier, CEM (V) | 1950 |
Multiple reaction monitoring (MRM) transition pair | 304 > 258 and 304 > 276 |
Time (Min) | Radiochemical Purity (RCP, %) |
---|---|
0 | 95.76 |
5 | 93.28 |
10 | 91.07 |
30 | 87.46 |
60 | 85.68 |
120 | 79.95 |
240 | 72.13 |
%ID/g | 5 min | 10 min | 30 min | 60 min | 120 min | 240 min | |
---|---|---|---|---|---|---|---|
Cerebellum | 2.22 ± 0.12 | 0.74 ± 0.04 | 0.07 ± 0.00 | 0.02 ± 0.00 | 0.01 ± 0.00 | 0.00 ± 0.00 | |
Pons/Medulla | 1.80 ± 0.19 | 0.60 ± 0.06 | 0.06 ± 0.01 | 0.02 ± 0.00 | 0.01 ± 0.00 | 0.00 ± 0.00 | |
Striatum | 3.27 ± 0.32 | 1.09 ± 0.11 | 0.11 ±0.01 | 0.04 ± 0.00 | 0.02 ± 0.00 | 0.01 ± 0.00 | |
Cortex (Amyglada) | 5.28 ± 0.60 | 2.16 ± 0.39 | 0.38 ± 0.02 | 0.06 ± 0.01 | 0.03 ± 0.00 | 0.01 ± 0.00 | |
Hippocampus | 4.57 ± 0.56 | 1.82 ± 0.19 | 0.25 ± 0.01 | 0.05 ± 0.00 | 0.03 ± 0.00 | 0.01 ± 0.00 | |
Thalamus | 4.22 ± 0.41 | 1.61 ± 0.14 | 0.14 ± 0.00 | 0.05 ± 0.00 | 0.02 ± 0.00 | 0.01 ± 0.00 | |
Hypothalamus | 3.71 ± 0.18 | 1.34 ± 0.06 | 0.12 ± 0.00 | 0.04 ± 0.00 | 0.02 ± 0.00 | 0.01 ± 0.00 | |
SBR | 5 min | 10 min | 30 min | 60 min | 120 min | 240 min | |
Cerebellum | 0.25 ± 0.18 | 0.36 ± 0.26 | 0.16 ± 0.15 | 0.36 ± 0.14 | 0.32 ± 0.07 | 0.71 ± 0.32 | |
Striatum | 0.84 ± 0.37 | 0.92 ± 0.34 | 0.91 ±0.29 | 1.00 ± 0.36 | 0.94 ± 0.24 | 0.98 ± 0.33 | |
Cortex (Amyglada) | 1.96 ± 0.49 | 2.65 ± 0.85 | 5.32 ± 0.82 | 2.22 ± 0.42 | 2.14 ± 0.43 | 1.71 ± 0.51 | |
Hippocampus | 1.55 ± 0.37 | 2.06 ± 0.41 | 3.24 ± 0.52 | 1.78 ± 0.32 | 1.71 ± 0.41 | 1.73 ± 0.87 | |
Thalamus | 1.37 ± 0.37 | 1.70 ± 0.40 | 1.37 ± 0.36 | 1.57 ± 0.31 | 1.51 ± 0.30 | 1.53 ± 0.58 | |
Hypothalamus | 1.08 ± 0.26 | 1.25 ± 0.28 | 1.15 ± 0.14 | 1.26 ± 0.21 | 1.20 ± 0.19 | 1.02 ± 0.42 |
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Chen, W.-H.; Chiu, C.-H.; Farn, S.-S.; Cheng, K.-H.; Huang, Y.-R.; Lee, S.-Y.; Fang, Y.-C.; Lin, Y.-H.; Chang, K.-W. Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging. Pharmaceuticals 2023, 16, 764. https://doi.org/10.3390/ph16050764
Chen W-H, Chiu C-H, Farn S-S, Cheng K-H, Huang Y-R, Lee S-Y, Fang Y-C, Lin Y-H, Chang K-W. Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging. Pharmaceuticals. 2023; 16(5):764. https://doi.org/10.3390/ph16050764
Chicago/Turabian StyleChen, Wei-Hsi, Chuang-Hsin Chiu, Shiou-Shiow Farn, Kai-Hung Cheng, Yuan-Ruei Huang, Shih-Ying Lee, Yao-Ching Fang, Yu-Hua Lin, and Kang-Wei Chang. 2023. "Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging" Pharmaceuticals 16, no. 5: 764. https://doi.org/10.3390/ph16050764
APA StyleChen, W. -H., Chiu, C. -H., Farn, S. -S., Cheng, K. -H., Huang, Y. -R., Lee, S. -Y., Fang, Y. -C., Lin, Y. -H., & Chang, K. -W. (2023). Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging. Pharmaceuticals, 16(5), 764. https://doi.org/10.3390/ph16050764