Kim Jaeah, Basiri Babak, Hassan Chopie, Punt Carine, van der Hage Erik, den Besten Cathaline, Bartlett Michael G
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA.
ProQR Therapeutics N.V., Leiden, the Netherlands.
Mol Ther Nucleic Acids. 2019 Sep 6;17:714-725. doi: 10.1016/j.omtn.2019.07.006. Epub 2019 Jul 22.
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.
依鲁福生（曾用名QR-010）是一种33聚体的2'-O-甲基修饰硫代磷酸反义寡核苷酸，靶向囊性纤维化患者编码CFTR蛋白的基因中的F508del突变。在本研究中，将依鲁福生与内切酶、外切酶和小鼠肝脏匀浆孵育，以阐明其体外代谢情况。使用小鼠和猴子来确定吸入依鲁福生后在体内肝脏和肺部的代谢情况。我们开发了一种液相色谱-质谱方法，用于鉴定和半定量依鲁福生的代谢产物，然后将该方法应用于体外和体内代谢研究。在蛋白酶K消化后，采用固相萃取法进行样品制备。在体外孵育系统的小鼠肝脏中，以及在体内小鼠和猴子研究的肝脏和肺组织样本中，均观察到依鲁福生经3'外切酶作用产生的链缩短代谢产物。本研究为在体外和体内研究中进一步表征2'-核糖修饰硫代磷酸寡核苷酸的代谢产物提供了方法，以支持寡核苷酸疗法的开发。
