In vitro metabolism of 2′‐ribose unmodified and modified phosphorothioate oligonucleotide therapeutics using liquid chromatography mass spectrometry

Abstract

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.

Safety Testing of an Antisense Oligonucleotide Intended for Pediatric Indications in the Juvenile Göttingen Minipig, including an Evaluation of the Ontogeny of Key Nucleases

“…However, this does not fit with any of the 3′-exonucleases’ expression patterns. As ASO gapmers are protected at the 3′- and 5′-end from exonuclease degradation by the additional flank modifications, they are known to be initially cleaved by endonucleases in the tissue [ 69 , 70 , 116 ] of different species [ 16 , 50 ], leading to short fragments that exonucleases may degrade further. As such, this supports the correlation between liver nuclease activity and endonuclease expression pattern, indicating that the nuclease activity in the liver of minipigs initially proceeds through endonucleases.…”

Novel advances in biotransformation and bioactivation research – 2020 year in review

“…In the work by Kim et al (2020) , unmodified (PO backbone), first-generation (PS backbone), and second-generation (PS backbone/2′-MOE gapmer) ASOs were investigated for their metabolic stability in in vitro systems including endo/exonucleases, mouse liver homogenate and human liver microsomes. After incubation, the samples were prepared by solid phase extraction and analyzed using ion pair reversed phase liquid chromatography and high-resolution time of flight mass spectrometry (SYNAPT G2) under negative mode.…”

Bioanalysis and Biotransformation of Oligonucleotide Therapeutics by Liquid Chromatography‐mass Spectrometry

“…This originally involved creating methoxy (OMe) derivatives but later evolved to MOE and LNAs including the methylated analog of LNAs which is referred to as a constrained ethyl modification (Fig. 2a) (Wilson & Keefe, 2006; Campbell & Wengel, 2011; Juliano, 2016; Kim, El Zahar, & Bartlett, 2020). While these modifications are quite beneficial in terms of increasing the stability of the oligonucleotides, they are disruptive to the activity of RNase H and prevent its typical cleavage of DNA‐RNA hybrids.…”

“…Gapmers have a complementary DNA sequence flanked by 3‐5 2′‐modified nucleotides on either side of the unmodified DNA sequence (Fig. 2b) (Kim, El Zahar, & Bartlett, 2020). One other modification of the sugar that can occur is the substitution of a morpholino group for the ribose.…”

“…( a ) Structure of unmodified oligonucleotides and first‐, second‐, and third‐generation ASOs. Reprinted with permission from Kim et al (2020), Biomedical Chromatography . ( b ) Gapmers.…”