Aberrant RNA splicing contributes to many human diseases, and splice-switching antisense oligonucleotides are ideally suited as a therapeutic strategy to modulate splicing and restore normal gene expression. Nusinersen (Spinraza™) has revolutionized the treatment of spinal muscular atrophy. It is a splice-switching oligonucleotide (SSO) modified with 2'-O-methoxyethyl (MOE). Here, we evaluate a next-generation ribose modification, 2'-O-[2-(methylamino)-2-oxoethyl] (NMA), which enhances the pharmacological properties of SSOs. We identified a long-lasting NMA-modified human candidate SSO, salanersen, that is three to four-fold more potent than nusinersen in human SMN2 transgenic mice. To evaluate the generality of the NMA chemistry, we applied it to modulation of SCN1A exon 20N splicing, a therapeutic strategy for Dravet syndrome. An NMA-modified SSO is 3.5 -fold more potent than STK-001, a MOE-modified SSO currently in clinical trials. Our data establish the NMA chemistry as a broadly applicable ribose modification that markedly improves the pharmacological profile of SSOs, supporting its development as a next-generation platform for splicing modulation therapies.

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19–23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

The 14 th edition of the Workshop on Recent Issues in Bioanalysis (14 th WRIB) was held virtually on June 15–29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings.[Table: see text].

Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both humanand mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitros-amine- induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.

BioanalysisVol. 11, No. 21 EditorialBioanalysis considerations on the pharmacokinetic evaluation of antisense therapeuticsDaniel A Norris, Noah Post, Rosie Z Yu, Sarah Greenlee & Yanfeng WangDaniel A Norris *Author for correspondence: E-mail Address: dnorris@ionisph.comhttps://orcid.org/0000-0003-2074-5862Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA, Noah PostIonis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA, Rosie Z YuIonis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA, Sarah GreenleeIonis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA & Yanfeng WangIonis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USAPublished Online:25 Oct 2019https://doi.org/10.4155/bio-2019-0194AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: antisenseelectrochemiluminescenceELISALC–MSoligonucleotidespharmacokineticsReferences1. Ackermann EJ, Guo S, Benson MD et al. Suppressing transthyretin production in mice, monkeys and humans using 2nd-generation antisense oligonucleotides. Amyloid 23(3), 148–157 (2016).Crossref, Medline, CAS, Google Scholar2. Chiriboga CA, Swoboda KJ, Darras BT et al. Results from a Phase 1 study of nusinersen (ISIS-SMNRx) in children with spinal muscular atrophy. Neurology 86(10), 890–897 (2016).Crossref, Medline, CAS, Google Scholar3. Finkel RS, Chiriboga CA, Vajsar J et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a Phase II, open-label, dose-escalation study. Lancet 388(10063), 3017–3026 (2016).Crossref, Medline, CAS, Google Scholar4. Geary RS, Norris D, Yu R, Bennett CF. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug Deliv. Rev. 87, 46–51 (2015).Crossref, Medline, CAS, Google Scholar5. Tabrizi SJ, Leavitt BR, Landwehrmeyer GB et al. Targeting Huntingtin expression in patients with Huntington's disease. N. Engl. 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Toxicol. 15(6), 475–485 (2019).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByAntisense oligonucleotide targeting DMPK in patients with myotonic dystrophy type 1: a multicentre, randomised, dose-escalation, placebo-controlled, phase 1/2a trialThe Lancet Neurology, Vol. 22, No. 3Drug Metabolism and Pharmacokinetics of Antisense Oligonucleotide Therapeutics: Typical Profiles, Evaluation Approaches, and Points to Consider Compared with Small Molecule Drugs3 February 2023 | Nucleic Acid Therapeutics, Vol. 52A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples8 February 2023 | Molecules, Vol. 28, No. 4Non-invasive Imaging of Antisense Oligonucleotides in the Brain via In Vivo Click Chemistry2 June 2022 | Molecular Imaging and Biology, Vol. 24, No. 6Bioanalysis of Oligonucleotide by LC–MS: Effects of Ion Pairing Regents and Recent Advances in Ion-Pairing-Free Analytical Strategies7 December 2022 | International Journal of Molecular Sciences, Vol. 23, No. 24Microflow LC–MS/MS to improve sensitivity for antisense oligonucleotides bioanalysis: critical role of sample cleannessDi Jiang & Long Yuan10 January 2023 | Bioanalysis, Vol. 14, No. 21Development and multicenter validation of an LC–MS-based bioanalytical method for antisense therapeuticsYuchen Sun, Shin-ichiro Nitta, Kosuke Saito, Ryuta Hosogai, Keiko Nakai, Ryoya Goda, Hisao Shimizu, Hisashi Fujita, Masaaki Kakehi, Kazuyuki Murata, Takeru Yamaguchi, Takeshi Okuzono, Shinichi Yamane, Mitsuhiko Kawabata, Takayuki Matsunuma, Kentaro Takahara, Noriko Kato, Masaki Yamada, Tokuyuki Yoshida, Takao Inoue & Yoshiro Saito21 November 2022 | Bioanalysis, Vol. 14, No. 18Validation and application of hybridization liquid chromatography-tandem mass spectrometry methods for quantitative bioanalysis of antisense oligonucleotidesPei Li, Jean-François Dupuis, Valerie Vrionis, Kevork Mekhssian, Tom Magee & Long Yuan12 May 2022 | Bioanalysis, Vol. 14, No. 9High-sensitivity quantification of antisense oligonucleotides for pharmacokinetic characterizationShilpi Mahajan, Hui Zhao, Kristina Kovacina, Eric Lachacz, Sany Hoxha, Josolyn Chan & Meina Liang17 May 2022 | Bioanalysis, Vol. 14, No. 9miR-221/222 as biomarkers and targets for therapeutic intervention on cancer and other diseases: A systematic reviewMolecular Therapy - Nucleic Acids, Vol. 27Recent advances in therapeutic nucleic acids and their analytical methodsJournal of Pharmaceutical and Biomedical Analysis, Vol. 206Distribution and biotransformation of therapeutic antisense oligonucleotides and conjugatesDrug Discovery Today, Vol. 26, No. 10Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometryYuchen Sun, Shin-ichiro Nitta, Kosuke Saito, Ryuta Hosogai, Keiko Nakai, Ryoya Goda, Masaaki Kakehi, Kazuyuki Murata, Takeru Yamaguchi, Takeshi Okuzono, Shinichi Yamane, Yoshimasa Enoki, Mitsuhiko Kawabata, Kentaro Takahara, Shimpei Sato, Tokuyuki Yoshida, Takao Inoue & Yoshiro Saito26 November 2020 | Bioanalysis, Vol. 12, No. 24In vivo and in vitro studies of antisense oligonucleotides – a review1 January 2020 | RSC Advances, Vol. 10, No. 57The exciting world of oligonucleotides: a multidisciplinary complex challenge for multitasking ingenious bioanalystsYuanxin Xu, Fabio Garofolo & Adrien Musuku12 December 2019 | Bioanalysis, Vol. 11, No. 21 Vol. 11, No. 21 Follow us on social media for the latest updates Metrics Downloaded 518 times History Received 31 July 2019 Accepted 7 October 2019 Published online 25 October 2019 Published in print November 2019 Information© 2019 Newlands PressKeywordsantisenseelectrochemiluminescenceELISALC–MSoligonucleotidespharmacokineticsFinancial & competing interests disclosureAll authors are employees of Ionis Pharmaceuticals. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Volanesorsen (previously known as ISIS 304801) is a 20-nucleotide partially 2′-O-(2-methoxyethyl) (2′-MOE)–modified antisense oligonucleotide (ASO) gapmer, which was recently approved in the European Union as a novel, first-in-class treatment in the reduction of triglyceride levels in patients with familial chylomicronemia syndrome. We characterized the absorption, distribution, metabolism, and excretion characteristics of volanesorsen in mice, rats, monkeys, and humans, in either radiolabeled or nonradiolabeled studies. This also included the characterization of all of the observed ASO metabolite species excreted in urine. Volanesorsen is highly bound to plasma proteins that are similar in mice, monkeys, and humans. In all species, plasma concentrations declined in a multiphasic fashion, characterized by a relatively fast initial distribution phase and then a much slower terminal elimination phase following subcutaneous bolus administration. The plasma metabolite profiles of volanesorsen are similar across species, with volanesorsen as the major component. Various shortened oligonucleotide metabolites (5–19 nucleotides long) were identified in tissues in the multiple-dose mouse and monkey studies, but fewer in the [3H]-volanesorsen rat study, likely due to a lower accumulation of metabolites following a single dose in rats. In urine, all metabolites identified in tissues were observed, consistent with both endo- and exonuclease-mediated metabolism and urinary excretion being the major elimination pathway for volanesorsen and its metabolites. Significance Statement We characterized the absorption, distribution, metabolism, and excretion (ADME) of volanesorsen, a partially 2′-MOE–modified antisense oligonucleotide, from mouse to man utilizing novel extraction and quantitation techniques in samples collected from preclinical toxicology studies, a 3H rat ADME study, and a phase 1 clinical trial.

Volanesorsen (previously known as ISIS 304801) is a 20-nucleotide partially 29-O-(2-methoxyethyl) (29-MOE)–modified antisense oligonucleotide (ASO) gapmer, which was recently approved in the European Union as a novel, first-in-class treatment in the reduction of triglyceride levels in patients with familial chylomicronemia syndrome. We characterized the absorption, distribution, metabolism, and excretion characteristics of volanesorsen in mice, rats, monkeys, and humans, in either radiolabeled or nonradiolabeled studies. This also included the characterization of all of the observed ASOmetabolite species excreted in urine. Volanesorsen is highly bound to plasma proteins that are similar in mice, monkeys, and humans. In all species, plasma concentrations declined in a multiphasic fashion, characterized by a relatively fast initial distribution phase and then a much slower terminal elimination phase following subcutaneous bolus administration. The plasma metabolite profiles of volanesorsen are similar across species, with volanesorsen as the major component. Various shortened oligonucleotide metabolites (5–19 nucleotides long) were identified in tissues in the multiple-dose mouse and monkey studies, but fewer in the [H]-volanesorsen rat study, likely due to a lower accumulation of metabolites following a single dose in rats. In urine, all metabolites identified in tissues were observed, consistent with both endoand exonucleasemediated metabolism and urinary excretion being the major elimination pathway for volanesorsen and its metabolites. SIGNIFICANCE STATEMENT We characterized the absorption, distribution, metabolism, and excretion (ADME) of volanesorsen, a partially 29-MOE–modified antisense oligonucleotide, from mouse to man utilizing novel extraction and quantitation techniques in samples collected from preclinical toxicology studies, a H rat ADME study, and a phase 1 clinical trial.

The 6-month Tg.rasH2 mouse carcinogenicity model provides an acceptable alternative to the 2-year carcinogenicity study in CD-1 mice. However, key questions related to the use of this model for testing antisense oligonucleotides (ASOs) include the similarity in the biologic response between mouse strains and the feasibility of using data from the CD-1 mouse to set doses and dose schedules for a Tg.rasH2 carcinogenicity study. To evaluate the potential strain differences, four distinct 2′-O-(2-methoxyethyl) ASOs were administered to CByB6F1 (wild type), Tg.rasH2 (hemizygous), and CD-1 mice. There were no meaningful differences in clinical signs, body weight, food consumption, or serum chemistry and hematology parameters. Histopathology evaluation indicated little to no difference in the spectrum or magnitude of changes present. The cytokine/chemokine response was also not appreciably different between the strains. This was consistent with the similarity in ASO concentration in the liver between the mouse strains tested. As the class effects of the ASOs were not meaningfully different between CD-1, CByB6F1, or Tg.rasH2 mice, data from nonclinical studies in CD-1 mice can be used for dose selection and expectation of effect in the Tg.rasH2 mouse.

Antisense oligonucleotides are metabolized by nucleases and drug interactions with small drug molecules at either the cytochrome P450 (CYP) enzyme or transporter levels have not been observed to date. Herein, a comprehensive in vitro assessment of the drug-drug interaction (DDI) potential was carried out with four 2'-O-(2-methoxyethyl)-modified antisense oligonucleotides (2'-MOE-ASOs), including a single triantennary N-acetyl galactosamine (GalNAc(3))-conjugated ASO. Several investigations to describe the DDI potential of a 2'-MOE-ASO conjugated to a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors are explored. The inhibition on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 and induction on CYP1A2, CYP2B6, and CYP3A4 were investigated in cryopreserved hepatocytes using up to 100 mu M of each ASO. No significant inhibition (half maximal inhibitory concentration [IC50] > 100 mu M) or induction was observed based on either enzymatic phenotype or mRNA levels. In addition, transporter interaction studies were conducted with nine major transporters per recommendations from regulatory guidances and included three hepatic uptake transporters, organic cation transporter 1 (OCT1), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3; three renal uptake transporters, organic anion transporter 1 (OAT1), OAT3, and OCT2; and three efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and bile salt export pump (BSEP). None of the four ASOs (10 mu M) were substrates of any of the nine transporters, with uptake <2-fold compared to controls, and efflux ratios were below 2.0 for BCRP and P-gp. Additionally, neither of the four ASOs showed meaningful inhibition on any of the nine transporters tested, with the mean percent inhibition ranging from -38.3% to 24.2% with 100 mu M ASO. Based on these findings, the unconjugated and GalNAc(3)-conjugated 2'-MOE-ASOs would have no or minimal DDI with small drug molecules via any major CYP enzyme or drug transporters at clinically relevant exposures.

Phosphorothioate (PS) modified antisense oligonucleotides (ASOs) have progressed rapidly in the clinic for treating a variety of disease indications. We previously demonstrated that the activity of PS ASOs in the liver can be enhanced by co-infusion of an excipient oligonucleotide (EON). It was posited that the EON saturates a nonproductive uptake pathway(s) thereby permitting accumulation of the PS ASO in a productive tissue compartment. In this report, we measured PS ASO activity following administration by bolus, infusion or co-fusion with EON within hepatocytes and nonparenchymal cells (NPCs), of the liver. This revealed that while ASOs accumulate preferentially in NPCs, they are intrinsically more active in hepatocytes. Furthermore, we show that the EON enhances ASO potency when infused up to 72h before or after administration of the active ASO suggesting that the EON can saturate and displace the ASO from nonproductive to productive compartments. Physical presence of the EON in tissues was required for optimal potentiation suggesting that there is a dynamic distribution of the ASO and EON between the compartments. Lastly, using a candidate approach, we confirmed Stabilin-2 as a molecular pathway for ASO uptake in sinusoidal endothelial cells and the ASGR as a pathway for ASO uptake into hepatocytes in the liver.

Triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency via receptor-mediated uptake. In the present study, the in vivo pharmacology of a 2'-O-(2-methoxyethyl)-modified ASO conjugated with GalNAc3 (ISIS 681257) together with its unmodified congener (ISIS 494372) targeting human apolipoprotein (a) (apo(a)), were studied in human LPA transgenic mice. Further, the disposition kinetics of ISIS 681257 was studied in CD-1 mice. ISIS 681257 demonstrated over 20-fold improvement in potency over ISIS 494372 as measured by liver apo(a) mRNA and plasma apo(a) protein levels. Following subcutaneous (SC) dosing, ISIS 681257 cleared rapidly from plasma and distributed to tissues. Intact ISIS 681257 was the major full-length oligonucleotide species in plasma. In tissues, however, GalNAc sugar moiety was rapidly metabolized and unconjugated ISIS 681257 accounted > 97% of the total exposure, which was then cleared slowly from tissues with a half-life of 7-8 days, similar to the half-life in plasma. ISIS 681257 is highly bound to plasma proteins (> 94% bound), which limited its urinary excretion. This study confirmed dose-dependent exposure to the parent drug ISIS 681257 in plasma and rapid conversion to unconjugated ASO in tissues. Safety data and the extended half-life support its further development and weekly dosing in phase 1 clinical studies.

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 H-3-radiolabeled (H-3 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 beta-position followed by dioxidation at the alpha-arm, and lastly, tri and tetra oxidations on the two remaining beta-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-beta-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.

Triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency due to receptor-mediated uptake into hepatocyte. The disposition and pharmacokinetics of ISIS 681257, a GalNAc3-conjugated ASO, were studied in monkeys. Following subcutaneous (SC) injection, ISIS 681257 was rapidly absorbed into the systemic circulation, with peak plasma levels observed within hours after dosing. After reaching Cmax, plasma concentrations rapidly declined in a multiexponential manner and were characterized by a dominant initial rapid distribution phase in which drug transferred to tissues from circulation, followed by a much slower terminal elimination phase (half-life of 4 weeks). Intact ISIS 681257 is the major full-length oligonucleotide species in plasma (≥70%). In tissues, the conjugated-GalNAc sugar moiety was rapidly metabolized, leaving the fully unconjugated form as the only full-length oligonucleotide detected at 48 h after dosing. Unconjugated ISIS 681257 cleared slowly from tissues with a half-life of 4 weeks. ISIS 681257 was highly bound to plasma proteins (>97% bound), which limited its urinary excretion. Disposition of ISIS 681257 in plasma and liver appeared nonlinear over the 1-40 mg/kg dose range studied. The plasma and liver tissue concentration data were well described by a population based mixed-effects modeling approach with Michaelis-Menten uptake from plasma to liver. Safety data from the study and the good exposure, as well as the extended half-life of the unconjugated ASO in the liver, support further development and less frequent dosing in Phase I clinical study.