Rapid assessment of metabolism and disposition of conjugated oligonucleotide vutrisiran in rats using a sensitive LC-HRMS platform

Vutrisiran is an N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA therapeutic approved for targeted hepatic delivery and long-acting gene silencing; however, detailed in vitro and in vivo metabolism data have not been published. In this study, a sensitive liquid chromatography coupled with high resolution mass spectrometry platform was established to systematically evaluate the metabolism and disposition of vutrisiran in rats after administration of a low, pharmacologically relevant dose (3 mg/kg). Vutrisiran exhibited high systemic metabolic stability, with the intact parent drug as the predominant species in plasma and urine. Tissue metabolite profiling revealed distinct organ-specific pathways, including hepatic GalNAc cleavage followed by slow 3'-exonuclease-mediated degradation, and a low-abundance but unique kidney-specific 5'-truncation of the sense strand (n-4). Renal excretion was the primary route for elimination of intact drug, whereas biliary excretion was responsible for clearance of extensively catabolized fragments. The overall excretion pattern was consistent with that reported for Other GalNAc-conjugated oligonucleotides. These rat metabolic profiling data provide insight into the disposition of vutrisiran in humans and represent the first reported in vivo disposition characterization of vutrisiran. The liquid chromatography coupled with high resolution mass spectrometry platform developed in this study enabled highly sensitive metabolite profiling and identification. A semiquantitative approach based on mass spectrometry peak area integration was validated by demonstrating close numerical agreement with UV detection. This platform supports metabolite profiling in plasma and target tissues for pharmacokinetics/pharmacodynamics studies and rapid disposition characterization in Animals and may serve as an alternative or pilot approach to inform the design and necessity of human radiolabeled mass-balance studies for oligonucleotide therapeutics. SIGNIFICANT STATEMENT: This work reports the first in vivo disposition assessment of vutrisiran using a sensitive liquid chromatography coupled with high resolution mass spectrometry platform for characterizing oligonucleotide metabolism and excretion at pharmacologically relevant doses. These findings advance the understanding of vutrisiran disposition and support the use of nonradiolabeled strategies for metabolite profiling and disposition evaluation of oligonucleotide therapeutics.