In Vitro-In Vivo Metabolite Profiling and Identification of Oligonucleotide

Oligonucleotides are synthetic single or double strands of modified RNA (ribonucleic acid) or DNA (deoxyribonucleic acid), less than 100 nt (nucleotides), which can be used to modulate gene expression by binding to RNA or DNA through Watson–Crick base pairing. They can, in theory, target any gene of interest since only the right nucleotide sequence along the targeted DNA or RNA needs to be selected, so, oligonucleotides should be much more specific than small molecule drugs. The characterization of oligonucleotide and any relevant metabolites had been listed in the considerations in FDA guidance (June 2022), As for all marketed oligonucleotide drugs, the materials for in vitro and in vivo metabolic studies were submitted for IND filings to clarify the metabolic behaviors and clearance pathways. Based on the HRMS technology and related software, a strategy has been set up for Oligo A in in vitro and in vivo metabolism studies to characterize the relevant metabolites and indicate the in vitro-in vivo metabolic correlation. Based on our investigation, the metabolite profiling of Oligo A in liver S9 fractions was more correlated with the metabolite profile in in vivo liver comparing to liver microsomes and hepatocytes. The metabolism of Oligo A was evaluated in vitro in mouse, rat, dog, monkey and human liver S9 fractions, and in vivo in monkey liver samples by UPLC-UV-HRMS.