Quantitative Determination of Click-Reactive Antisense Oligonucleotide and Its Reactivity in Rat Brains by Hybridization LC-MS/MS to Support Pretargeted PET Imaging
Di Jiang, Brendon E. Cook, Thomas Pickel, Long Yuan
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引用次数: 0
Abstract
Antisense oligonucleotide (ASO) therapeutics are rapidly increasing in prevalence for the treatment of neurodegenerative diseases. A novel pretargeted positron emission tomography (PET) imaging methodology based on in vivo click chemistry was developed recently to assess ASO biodistribution in the brain. To validate the performance of the method, a sensitive, accurate, and reliable method to quantitatively determine the reactivity and distribution of click-reactive ASO in the brain is required. A major challenge is that the click-reactive ASO may undergo oxidation or rearrangement in vivo and lose reactivity to the TCO on the radiotracer. With their small mass differences, it is difficult to differentiate these unreactive degradants from the intact reactive ASO by LC-MS/MS. Here, we developed a novel postextraction analyte derivatization methodology utilizing conjugation of the click-reactive ASO, Malat1 ASO with a 1,2,4,5-methyltetrazine (MeTz) modification (Malat1 ASO-MeTz), with a TCO ligand (TCO-PEG4-DBCO) to differentiate the reactive and unreactive ASOs. The intact and reactive form of Malat1 ASO-MeTz was determined by LC-MS/MS analysis of the conjugation reaction product. A hybridization LC-MS/MS method that simultaneously quantifies Malat1 ASO-MeTz and Malat1 ASO over the range of 1.00–1000 ng/mL was also developed to determine the ASO distribution in rat brains. The methods were successfully applied to a pretargeted PET imaging study in rats. This is the first report that a postextraction analyte derivatization methodology was successfully developed for determining the reactive form and reactivity of click-reactive ASOs. The developed bioanalytical methodology will also significantly facilitate the evaluation of other radiotracers for pretargeted PET imaging.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.