title: Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys.
abstract: Triantennary N-acetyl galactosamine (GalNAc3) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of (3)H-radiolabeled ((3)H placed in THA) or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5'-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.
authors.affiliation: Department of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Colby S
authors.initials: CS
authors.lastname: Shemesh
authors.affiliation: Department of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Rosie Z
authors.initials: RZ
authors.lastname: Yu
authors.affiliation: Structural Biology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Gaus
authors.affiliation: Department of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Greenlee
authors.affiliation: Department of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Noah
authors.initials: N
authors.lastname: Post
authors.affiliation: Structural Biology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Karsten
authors.initials: K
authors.lastname: Schmidt
authors.affiliation: Medicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Migawa
authors.affiliation: Medicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Punit P
authors.initials: PP
authors.lastname: Seth
authors.affiliation: Toxicology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Zanardi
authors.affiliation: Medicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.affiliation: Medicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Swayze
authors.affiliation: Toxicology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Henry
authors.affiliation: Department of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: Wang