Metabolite Profiling of the Antisense Oligonucleotide Eluforsen Using Liquid Chromatography-Mass Spectrometry

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.

Attachment of hybridizable oligonucleotides to a silica support and its application for selective extraction of unmodified and antisense oligonucleotides from serum samples

“…14 Based on our results and application of full-length complementary strand for the extraction we are convinced that such approach allows for effective extraction of n-1, n-2, n-3 and n-4 metabolites, however we suppose that high selectivity will be observed also for shorter metabolites. These effects are probably strictly connected with application of completely complementary strand bonded to solid surface, contrary to approach presented by Kim et al 14…”

“…Despite the fact that attachment of OGNs to a solid support is of interest for many biotechnological applications, these materials are not widespread used or the selective extraction of OGNs based on hybridization. Lately, Bartlett et al 14 have used streptavidin magnetic beads with biotinylated capture strand (5 0 -AAAGAAAATATCATCT-3 0 ) to selectively capture eluforsen and its metabolites. This drug is antisense oligonucleotide (ASO) so it is a modied (at phosphate group or sugar molecules) derivative of OGNs.…”

“…ASOs are used in antisense therapy, which is based on their binding to complementary fragments of nucleic acids, similarly to their hybridization with OGNs immobilized at solid supports. However, Bartlett et al 14 have concluded that their application is limited due to difficulties associated with the loss of affinity of a variety of metabolite from a capture strand despite their high specicity and good recovery (80-95%). On the other hand in another paper it was shown that OGNs probe provided very effective cross-hybridization with 3 0 n-1 and n-2 metabolites.…”

“…5,6 It was also shown that the sensitivity and specicity of the assay depend on the choice of the methods/reagents and the ONGs sequences. [14][15][16] On the other hand hybridization based techniques provides the best sensitivity and throughput compared with other methods. 5,6,[13][14][15][16] The limited application of hybridization based techniques for selective extraction is due to problems associated with the reproducibility, long-term stability, non-specic binding of DNA, special care during designing capture probes to ensure appropriate affinity for the OGN analyte.…”

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

“…Based on our previously optimized method using 15 m m DMCHA and 25 m m HFIP (Kim et al, 2019), a final mobile phase consisting of 30 m m DMCHA and 100 m m HFIP was utilized. The separation method was further improved significantly by optimizing the concentration of mobile phase modifiers.…”