In Vivo Metabolite Profiles of a GalNAc Conjugated Antisense Oligonucleotide AZD8233 Using Liquid Chromatography-High Resolution Mass Spectrometry: a Cross Species Comparison in Animals and Humans

AZD8233, a liver-targeting antisense oligonucleotide (ASO), inhibits subtilisin/kexin type 9 protein synthesis. It is a phosphorothioated 3-10-3 gapmer with a central DNA sequence flanked by constrained 2'- -ethyl 2',4'-bridged nucleic acid (cEt-BNA) wings and conjugated to a triantennary -acetylgalactosamine (GalNAc) ligand at the 5'-end. Herein we report the biotransformation of AZD8233, as given by liver, kidney, plasma and urine samples, following repeated subcutaneous administration to humans, mice, rats, rabbits and monkeys. Metabolite profiles were characterized using liquid chromatography high-resolution mass spectrometry. Metabolite formation was consistent across species, mainly comprising hydrolysis of GalNAc sugars, phosphodiester-linker hydrolysis releasing the full-length ASO, and endonuclease-mediated hydrolysis within the central DNA gap followed by exonuclease-mediated 5'- or 3'-degradation. All metabolites contained the 5'- or 3'-cEt-BNA terminus. Most shortmer metabolites had the free terminal alcohol at 5'- and 3'-positions of ribose, although six were found retaining the terminal 5'-phosphorothioate group. GalNAc conjugated shortmer metabolites were also observed in urine. Synthesized metabolite standards were applied for (semi-)quantitative metabolite assessment. Intact AZD8233 was the major component in plasma, whereas the unconjugated full-length ASO was predominant in tissues. In plasma, most metabolites were shortmers retaining the 3'-cEt-BNA terminus, whereas metabolites containing the 5'- or 3'-cEt-BNA terminus were detected in both tissues and urine. All metabolites in human plasma were also detected in all non-clinical species, and all human urine metabolites were detected in monkey urine. In general, metabolite profiles in animal species were qualitatively similar and quantitatively exceeded the exposures of the circulating metabolites in humans at the doses studied. This study presents metabolite identification and profiling of AZD8233, a GalNAc-conjugated ASO, in humans and animals. A biotransformation strategy for ASO drug candidates was established by utilizing tissue, plasma and urine samples collected from toxicology and/or clinical studies and LC-high-resolution mass spectrometry analysis without conducting bespoke radiolabeled ADME studies. The generated biotransformation package was considered adequate by health authorities to progress AZD8233 into a phase 3 programme, proving its applicability to future metabolism s