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Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry.
Mol Ther Nucleic Acids 2019; 17:714-725MT

Abstract

Eluforsen (previously known as QR-010) is a 33-mer 2'-O-methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3' exonuclease were observed in mouse liver in an in vitro incubation system and by either 3' exonuclease or 5' exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2'-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics.

Authors+Show Affiliations

Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA.Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA.ProQR Therapeutics N.V., Leiden, the Netherlands.ProQR Therapeutics N.V., Leiden, the Netherlands.ProQR Therapeutics N.V., Leiden, the Netherlands.ProQR Therapeutics N.V., Leiden, the Netherlands.Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA. Electronic address: mgbart@uga.edu.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31422288

Citation

Kim, Jaeah, et al. "Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry." Molecular Therapy. Nucleic Acids, vol. 17, 2019, pp. 714-725.
Kim J, Basiri B, Hassan C, et al. Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry. Mol Ther Nucleic Acids. 2019;17:714-725.
Kim, J., Basiri, B., Hassan, C., Punt, C., van der Hage, E., den Besten, C., & Bartlett, M. G. (2019). Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry. Molecular Therapy. Nucleic Acids, 17, pp. 714-725. doi:10.1016/j.omtn.2019.07.006.
Kim J, et al. Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry. Mol Ther Nucleic Acids. 2019 Sep 6;17:714-725. PubMed PMID: 31422288.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry. AU - Kim,Jaeah, AU - Basiri,Babak, AU - Hassan,Chopie, AU - Punt,Carine, AU - van der Hage,Erik, AU - den Besten,Cathaline, AU - Bartlett,Michael G, Y1 - 2019/07/22/ PY - 2019/01/18/received PY - 2019/07/10/revised PY - 2019/07/10/accepted PY - 2019/8/20/pubmed PY - 2019/8/20/medline PY - 2019/8/19/entrez KW - antisense oligonucleotide KW - cystic fibrosis KW - eluforsen KW - ion-pair KW - liquid chromatography KW - mass spectrometry KW - metabolism SP - 714 EP - 725 JF - Molecular therapy. Nucleic acids JO - Mol Ther Nucleic Acids VL - 17 N2 - Eluforsen (previously known as QR-010) is a 33-mer 2'-O-methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3' exonuclease were observed in mouse liver in an in vitro incubation system and by either 3' exonuclease or 5' exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2'-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics. SN - 2162-2531 UR - https://www.unboundmedicine.com/medline/citation/31422288/Metabolite_Profiling_of_the_Antisense_Oligonucleotide_Eluforsen_Using_Liquid_Chromatography-Mass_Spectrometry L2 - https://linkinghub.elsevier.com/retrieve/pii/S2162-2531(19)30196-9 DB - PRIME DP - Unbound Medicine ER -