Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys
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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2016-01-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S216225311730046X |
| _version_ | 1811216671015174144 |
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| author | Colby S Shemesh Rosie Z Yu Hans J Gaus Sarah Greenlee Noah Post Karsten Schmidt Michael T Migawa Punit P Seth Thomas A Zanardi Thazha P Prakash Eric E Swayze Scott P Henry Yanfeng Wang |
| author_facet | Colby S Shemesh Rosie Z Yu Hans J Gaus Sarah Greenlee Noah Post Karsten Schmidt Michael T Migawa Punit P Seth Thomas A Zanardi Thazha P Prakash Eric E Swayze Scott P Henry Yanfeng Wang |
| author_sort | Colby S Shemesh |
| collection | DOAJ |
| description | 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 3H-radiolabeled (3H 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. |
| first_indexed | 2024-04-12T06:42:17Z |
| format | Article |
| id | doaj.art-1de8c3f8738147bdba5db5c61c7a342f |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-04-12T06:42:17Z |
| publishDate | 2016-01-01 |
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| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-1de8c3f8738147bdba5db5c61c7a342f2022-12-22T03:43:40ZengElsevierMolecular Therapy: Nucleic Acids2162-25312016-01-015C10.1038/mtna.2016.31Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and MonkeysColby S Shemesh0Rosie Z Yu1Hans J Gaus2Sarah Greenlee3Noah Post4Karsten Schmidt5Michael T Migawa6Punit P Seth7Thomas A Zanardi8Thazha P Prakash9Eric E Swayze10Scott P Henry11Yanfeng Wang12Department of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USADepartment of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USAStructural Biology, IONIS Pharmaceuticals, Carlsbad, California, USADepartment of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USADepartment of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USAStructural Biology, IONIS Pharmaceuticals, Carlsbad, California, USAMedicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USAMedicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USAToxicology, IONIS Pharmaceuticals, Carlsbad, California, USAMedicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USAMedicinal Chemistry, IONIS Pharmaceuticals, Carlsbad, California, USAToxicology, IONIS Pharmaceuticals, Carlsbad, California, USADepartment of Pharmacokinetics and Clinical Pharmacology, IONIS Pharmaceuticals, Carlsbad, California, USATriantennary 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 3H-radiolabeled (3H 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.http://www.sciencedirect.com/science/article/pii/S216225311730046Xantisense oligonucleotidebiotransformationmetabolismN-acetyl galactosaminetargeted drug delivery |
| spellingShingle | Colby S Shemesh Rosie Z Yu Hans J Gaus Sarah Greenlee Noah Post Karsten Schmidt Michael T Migawa Punit P Seth Thomas A Zanardi Thazha P Prakash Eric E Swayze Scott P Henry Yanfeng Wang Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys Molecular Therapy: Nucleic Acids antisense oligonucleotide biotransformation metabolism N-acetyl galactosamine targeted drug delivery |
| title | Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys |
| title_full | Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys |
| title_fullStr | Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys |
| title_full_unstemmed | Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys |
| title_short | Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys |
| title_sort | elucidation of the biotransformation pathways of a galnac3 conjugated antisense oligonucleotide in rats and monkeys |
| topic | antisense oligonucleotide biotransformation metabolism N-acetyl galactosamine targeted drug delivery |
| url | http://www.sciencedirect.com/science/article/pii/S216225311730046X |
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