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Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys

Triantennary N-acetyl galactosamine (GalNAc(3)) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc(3) via a trishexylamino (THA)-C6 cluster significantly enhances antisense oligonucleotide (ASO) potency. Herein, the biotransformation, disposition,...

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Autores principales: Shemesh, Colby S, Yu, Rosie Z, Gaus, Hans J, Greenlee, Sarah, Post, Noah, Schmidt, Karsten, Migawa, Michael T, Seth, Punit P, Zanardi, Thomas A, Prakash, Thazha P, Swayze, Eric E, Henry, Scott P, Wang, Yanfeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014515/
https://www.ncbi.nlm.nih.gov/pubmed/27164023
http://dx.doi.org/10.1038/mtna.2016.31
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author Shemesh, Colby S
Yu, Rosie Z
Gaus, Hans J
Greenlee, Sarah
Post, Noah
Schmidt, Karsten
Migawa, Michael T
Seth, Punit P
Zanardi, Thomas A
Prakash, Thazha P
Swayze, Eric E
Henry, Scott P
Wang, Yanfeng
author_facet Shemesh, Colby S
Yu, Rosie Z
Gaus, Hans J
Greenlee, Sarah
Post, Noah
Schmidt, Karsten
Migawa, Michael T
Seth, Punit P
Zanardi, Thomas A
Prakash, Thazha P
Swayze, Eric E
Henry, Scott P
Wang, Yanfeng
author_sort Shemesh, Colby S
collection PubMed
description Triantennary N-acetyl galactosamine (GalNAc(3)) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc(3) 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 GalNAc(3)-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. GalNAc(3)-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc(3) 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 GalNAc(3)-conjugated-ASO metabolism pathways which facilitate similar development programs.
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spelling pubmed-50145152016-09-19 Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys Shemesh, Colby S Yu, Rosie Z Gaus, Hans J Greenlee, Sarah Post, Noah Schmidt, Karsten Migawa, Michael T Seth, Punit P Zanardi, Thomas A Prakash, Thazha P Swayze, Eric E Henry, Scott P Wang, Yanfeng Mol Ther Nucleic Acids Original Article Triantennary N-acetyl galactosamine (GalNAc(3)) is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc(3) 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 GalNAc(3)-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. GalNAc(3)-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc(3) 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 GalNAc(3)-conjugated-ASO metabolism pathways which facilitate similar development programs. Nature Publishing Group 2016-05 2016-05-10 /pmc/articles/PMC5014515/ /pubmed/27164023 http://dx.doi.org/10.1038/mtna.2016.31 Text en Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Original Article
Shemesh, Colby S
Yu, Rosie Z
Gaus, Hans J
Greenlee, Sarah
Post, Noah
Schmidt, Karsten
Migawa, Michael T
Seth, Punit P
Zanardi, Thomas A
Prakash, Thazha P
Swayze, Eric E
Henry, Scott P
Wang, Yanfeng
Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys
title Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys
title_full Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys
title_fullStr Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys
title_full_unstemmed Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys
title_short Elucidation of the Biotransformation Pathways of a Galnac(3)-conjugated Antisense Oligonucleotide in Rats and Monkeys
title_sort elucidation of the biotransformation pathways of a galnac(3)-conjugated antisense oligonucleotide in rats and monkeys
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014515/
https://www.ncbi.nlm.nih.gov/pubmed/27164023
http://dx.doi.org/10.1038/mtna.2016.31
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