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Title: To D or not to D – an in vitro/mass spectrometry screening method to confirm improved metabolic stability in deuterium-labelled radiotracers
Authors: Murphy, RB
Wyatt, NA
Fraser, BH
Yepuri, NR
Holden, PJ
Wotherspoon, ATL
Darwish, TA
Keywords: Spectroscopy
Labelled compounds
Tracer techniques
Issue Date: 3-Sep-2019
Publisher: Australian Nuclear Science and Technology Organisation
Citation: Murphy, R., Wyatt, N., Fraser, B., Yepuri, N., Holden, P., Wotherspoon, A. & Darwish, T. (2019). To D or not to D – an in vitro/mass spectrometry screening method to confirm improved metabolic stability in deuterium-labelled radiotracers. Paper presented at the ANSTO Young Researchers' Conference, Lucas Heights, NSW, Australia; 3 Sep 2019. Retrieved from:
Abstract: Deuteration, where hydrogen within molecules is switched with the non-radioactive and naturally occurring isotope deuterium, can lead to enhanced material properties. For example, the deuterium kinetic isotope effect is well known to improve the metabolic stability of molecules such as drugs, as the C–D bond is stronger and more difficult for enzymes to break than the C–H bond. However, the specific molecular location to deuterate to gain a metabolically favourable outcome may not be clear, without undertaking separate assays of both the deuterated and non-deuterated molecules, followed by separate HPLC-UV/radiometric measurement. In the case of compounds which also contain a radiolabel (e.g. radiotracers for diagnostic medical imaging), specialist infrastructure and teams are required, and chemical synthesis and bioanalysis are time-critical. Our ongoing work with the radiotracer [18F]PBR111, a TSPO ligand showing potential for imaging neuroinflammation1,2,3, demonstrated that [18F]PBR111-d4 (where d4 is incorporated at a specific site) has slower metabolic breakdown4,5 and a decreased rate of formation of polar metabolites in vitro (rat and human liver microsomes) relative to non-deuterated 5,6. Our recently published MS/MS method4 demonstrates the relative difference in metabolic stability without radiolabelling, by analysing different time points from a liver microsome assay which has been administered a 50:50 mixture of deuterated/non-deuterated compound. Pharmacokinetic parameters can also be determined from the data. Deuteration adjacent to the fluoro-alkyl group in PBR111 showed ~50% improvement in the stability of the intact radiotracer relative to non-deuterated using a ratio determination of the analogous MS transitions unique to the deuterated/non-deuterated compounds4. As a control, a second deuterated analogue was also synthesised4 with deuterium purposely incorporated at a site significantly less metabolised than the first site5, which our newly developed method was also able to confirm. We expect this simple and rapid method could be applied to deuterated and non-deuterated analogues of other biologically important molecules to determine the suitability of the chosen site of deuteration. For radiotracers, there is no requirement to radiolabel until studies progress to in vivo PET imaging.
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