In vitro metabolism of 2'-ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry.

Autor: Kim J; Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA, USA., El Zahar NM; Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA, USA.; Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt., Bartlett MG; Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA, USA.
Jazyk: angličtina
Zdroj: Biomedical chromatography : BMC [Biomed Chromatogr] 2020 Jul; Vol. 34 (7), pp. e4839. Date of Electronic Publication: 2020 May 07.
DOI: 10.1002/bmc.4839
Abstrakt: Antisense oligonucleotides (ASOs) have been touted as an emerging therapeutic class to treat genetic disorders and infections. The evaluation of metabolic stability of ASOs during biotransformation is critical due to concerns regarding drug safety. Because the effects of the modifications in ASOs on their metabolic stabilities are different from unmodified ASOs, studies that afford an understanding of these effects as well as propose proper methods to determine modified and unmodified ASO metabolites are imperative. An LC-tandem mass spectrometry method offering good selectivity with a high-quality separation using 30 mm N,N-dimethylcyclohexylamine and 100 mm 1,1,1,3,3,3-hexafluoro-2-propanol was utilized to identify each oligonucleotide metabolite. Subsequently, the method was successfully applied to a variety of in vitro systems including endo/exonuclease digestion, mouse liver homogenates, and then liver microsomes, after which the metabolic stability of unmodified versus modified ASOs was compared. Typical patterns of chain-shortened metabolites generated by mainly 3'-exonucleases were observed in phosphodiester and phosphorothioate ASOs, and endonuclease activity was identically observed in gapmers that showed relatively more resistance to nuclease degradation. Overall, the degradation of each ASO occurred more slowly corresponding to the degree of chemical modifications, while 5'-exonuclease activities were only observed in gapmers incubated in mouse liver homogenates. Our findings provide further understanding of the impact of modifications on the metabolic stability of ASOs, which facilitates the development of future ASO therapeutics.
(© 2020 John Wiley & Sons, Ltd.)
Databáze: MEDLINE
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