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.

中文翻译：

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使用液相色谱质谱法对2'-核糖未修饰和修饰的硫代磷酸酯寡核苷酸治疗剂进行体外代谢。

反义寡核苷酸（ASO）被吹捧为治疗遗传疾病和感染的新兴治疗类别。由于对药物安全性的关注，评估ASO在生物转化过程中的代谢稳定性至关重要。由于ASO修饰对它们的代谢稳定性的影响与未修饰的ASO不同，因此，有必要对这些影响进行理解并提出确定修饰和未修饰的ASO代谢物的适当方法的研究。使用30 m m N，N-二甲基环己胺和100 m m的LC串联质谱方法具有良好的选择性和高质量的分离效果1,1,1,3,3,3-六氟-2-丙醇用于鉴定每种寡核苷酸代谢产物。随后，该方法成功地应用于多种体外系统，包括内切/核酸外切酶消化，小鼠肝匀浆，然后是肝微粒体，然后比较未修饰和修饰的ASO的代谢稳定性。在磷酸二酯和硫代磷酸酯ASO中观察到主要由3'-核酸外切酶产生的典型的链缩短代谢产物，在缺口聚体中观察到的核酸内切酶活性相同，对核酸酶降解的抵抗力更大。总体而言，每种ASO的降解与化学修饰的程度相对较慢，而5'-核酸外切酶活性仅在小鼠肝匀浆中孵育的间隔体中观察到。我们的发现提供了对修饰对ASO代谢稳定性的影响的进一步理解，这有助于未来ASO治疗剂的开发。