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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.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with this NEW Regulation" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th 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 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication covers the recommendations on Mass Spectrometry Assays, Regulated Bioanalysis/BMV (Part 1A) and Regulatory Inputs (Part 1B). Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 7 and 8 (2024), respectively.
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Proteómica , Humanos , Proteómica/métodos , Espectrometría de Masas/métodos , Biomarcadores/análisis , Estados Unidos , Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Genética , Cromatografía/métodos , BlancoRESUMEN
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.
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Cromatografía , Vacunas , Biomarcadores , Tratamiento Basado en Trasplante de Células y Tejidos , Espectrometría de Masas , Oligonucleótidos , TecnologíaRESUMEN
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.
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Vesículas Extracelulares , Vacunas , Biomarcadores/análisis , Tratamiento Basado en Trasplante de Células y Tejidos , Vesículas Extracelulares/química , Humanos , Espectrometría de Masas/métodos , NanomedicinaRESUMEN
The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th 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 14th 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.
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Bioensayo/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Terapia Genética/métodos , Espectrometría de Masas/métodos , Historia del Siglo XXI , HumanosRESUMEN
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.
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Autofagia/fisiología , Oligonucleótidos Antisentido/metabolismo , Animales , Autofagosomas/metabolismo , Transporte Biológico/fisiología , Células Cultivadas , Endocitosis/fisiología , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/genética , Interferencia de ARN , Transducción de SeñalRESUMEN
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 human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-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.
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Acetilgalactosamina/química , Técnicas de Transferencia de Gen , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química , Animales , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Células Cultivadas , Expresión Génica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , RatonesRESUMEN
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.
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Apolipoproteína C-III/antagonistas & inhibidores , Proteínas Sanguíneas/metabolismo , Oligonucleótidos/farmacocinética , Adulto , Animales , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Voluntarios Sanos , Humanos , Hiperlipoproteinemia Tipo I/sangre , Hiperlipoproteinemia Tipo I/tratamiento farmacológico , Hiperlipoproteinemia Tipo I/genética , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Tasa de Depuración Metabólica , Ratones , Persona de Mediana Edad , Mutación , Oligonucleótidos/administración & dosificación , Ratas , Eliminación Renal , Especificidad de la Especie , Distribución Tisular , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
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.
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Carcinógenos/toxicidad , Genes ras , Oligonucleótidos Antisentido/toxicidad , Oligorribonucleótidos/toxicidad , Pruebas de Toxicidad , Animales , Secuencia de Bases , Carcinógenos/clasificación , Carcinógenos/farmacocinética , Citocinas/sangre , Femenino , Hemicigoto , Masculino , Ratones Endogámicos ICR , Ratones Transgénicos , Oligonucleótidos Antisentido/clasificación , Oligonucleótidos Antisentido/farmacocinética , Oligorribonucleótidos/clasificación , Oligorribonucleótidos/farmacocinética , Tamaño de los Órganos/efectos de los fármacos , Especificidad de Órganos , Especificidad de la Especie , Factores de Tiempo , Distribución Tisular , Pruebas de Toxicidad/métodos , Pruebas de Toxicidad/normasRESUMEN
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 (GalNAc3)-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 µM of each ASO. No significant inhibition (half maximal inhibitory concentration [IC50] > 100 µM) 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 µM) 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 µM ASO. Based on these findings, the unconjugated and GalNAc3-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.
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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 72 h 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.
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Excipientes/farmacocinética , Hígado/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/farmacocinética , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Excipientes/administración & dosificación , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/citología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Fosforotioatos/administración & dosificación , Distribución TisularRESUMEN
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.
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Acetilgalactosamina/farmacocinética , Glicoconjugados/farmacocinética , Lipoproteína(a)/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/farmacocinética , Acetilgalactosamina/metabolismo , Animales , Biotransformación , Proteínas Sanguíneas/metabolismo , Femenino , Glicoconjugados/metabolismo , Semivida , Hepatocitos/metabolismo , Inyecciones Subcutáneas , Hígado/metabolismo , Masculino , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Unión Proteica , División del ARNRESUMEN
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 ß-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining ß-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-ß-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.
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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.
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A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to extract and quantify the androgen concentration in the rat prostate. This method introduced a novel 96-well plate format for the extraction and derivatization of testosterone (T) and dihydrotestosterone (DHT) from rat prostatic tissue that greatly simplified the sample preparation procedure. Due to the difficulty to obtain reproducible specimens with non-detectable level of androgen, a matrix-free standard solution was used for method validation. Both T and DHT calibration curves were linear over the calibration range (12.5-2500 pg) with correlation coefficient values greater than 0.9900. The intra-day and inter-day accuracy, reported as %bias, and precision, reported as %CV, of T and DHT were within +/-10%. The lower limit of detection (LLOD) and lower limits of quantification (LLOQ) for both T and DHT were determined to be 5 and 12.5 pg. The validation results demonstrated the selectivity, sensitivity, accuracy, precision, linearity and ruggedness of the method, as well as the suitability of the method for simultaneous detection of T and DHT in rat prostatic tissues. The validated method was successfully applied to determine the physiological T and DHT level in rat prostatic tissues. Similarly to the serum concentration profile pattern, T and DHT intraprostatic levels peaked 2 h after lights-on and decreased after lights-off with DHT level approximately 4-fold greater than T.
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Cromatografía Liquida/métodos , Dihidrotestosterona/análisis , Próstata/metabolismo , Espectrometría de Masas en Tándem/métodos , Testosterona/análisis , Animales , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
Thioredoxin-interacting protein (Txnip) inhibits thioredoxin NADPH-dependent reduction of protein disulfides. Total Txnip knockout (TKO) mice adapted inappropriately to prolonged fasting by shifting fuel dependence of skeletal muscle and heart from fat and ketone bodies to glucose. TKO mice exhibited increased Akt signaling, insulin sensitivity, and glycolysis in oxidative tissues (skeletal muscle and hearts) but not in lipogenic tissues (liver and adipose tissue). The selective activation of Akt in skeletal muscle and hearts was associated with impaired mitochondrial fuel oxidation and the accumulation of oxidized (inactive) PTEN, whose activity depends on reduction of two critical cysteine residues. Whereas muscle- and heart-specific Txnip knockout mice recapitulated the metabolic phenotype exhibited by TKO mice, liver-specific Txnip knockout mice were similar to WT mice. Embryonic fibroblasts derived from knockout mice also accumulated oxidized (inactive) PTEN and had elevated Akt phosphorylation. In addition, they had faster growth rates and increased dependence on anaerobic glycolysis due to impaired mitochondrial fuel oxidation, and they were resistant to doxorubicin-facilitated respiration-dependent apoptosis. In the absence of Txnip, oxidative inactivation of PTEN and subsequent activation of Akt attenuated mitochondrial respiration, resulting in the accumulation of NADH, a competitive inhibitor of thioredoxin NADPH-reductive activation of PTEN. These findings indicate that, in nonlipogenic tissues, Txnip is required to maintain sufficient thioredoxin NADPH activity to reductively reactivate oxidized PTEN and oppose Akt downstream signaling.