Turbo Fire Schedule Template Week 9 10 11 12
Turbo Fire Schedule Template Week 9 10 11 12 – Non-clinical disposition and pharmacokinetic/pharmacodynamic properties of N-acetylgalactosamine-conjugated small interfering RNA are highly predictable and increase confidence in translation to humans
Robin McDougall, Diane Ramsden, Sagar Agarwal, Saket Agarwal, Krishna Aluri, Michael Arciprete, Christopher Brown, Elena Castellanos-Rizaldos, Klaus Charisse, Saeho Chong, Joseph Cichocki, Kevin Fitzgerald, Varunaria tem Ja Ruumaria Guther, Vashav Maje, Muthusamy Jayaraman, Jeffrey Kurz, Jing Li, Ju Liu, Xiumin Liu, Steven Liou, Chris Maclauchlin, Martin Maier, Muthiah Manoharan, Jayaprakash K. Nair, Christ, Gabriel Robbie, Peter The schmid Smith Akshay Vaishnaw, Scottin X, Yuanx , Xuemei Zhang, Ivan Zlatev, and Jing-Tao Wu
Turbo Fire Schedule Template Week 9 10 11 12
Acetylgalactosamine (GalNAc) ligands have become an important strategy for hepatocyte-targeted delivery, and with the recent approval of GIVLAR (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inglisiran) for the treatment of hypercholesterolemia, this technology is clinically well validated. Although much knowledge has been gained over decades of development, there is little published literature on the metabolism and pharmacokinetic properties of GalNAc-siRNA drugs. With this in mind, this mini-review aims to present a pooled analysis of these non-clinical absorption, distribution, metabolism and excretion (ADME) data to provide confidence in the transferability of these properties to humans. After subcutaneous administration, GalNAc-conjugated siRNAs are rapidly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination from the circulation via asialoglycoprotein receptor-mediated uptake into hepatocytes. These studies confirm that liver PK, including half-life and, more importantly, siRNA levels in the RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were performed to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies show that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, largely predictable, and can be accurately scaled to humans, allowing us to identify effective and safe clinical dosing regimens. in the absence of human liver. PK profiles.
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SIGNIFICANCE Several non-clinical ADME studies have been conducted to provide comprehensive insight into the disposition and elimination and cross-species pharmacokinetic/pharmacodynamic translation of GalNAc-conjugated siRNAs. These studies show that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, increasing confidence in the ability to extrapolate to humans.
RNA interference (RNAi) is an intrinsic mechanism of post-transcriptional gene silencing mediated by double-stranded small oligonucleotides of 21–25 nucleotides in length called short interfering RNA (siRNA) (Fire et al., 1998; Meister and Tuschl, 2004 ). This catalytic process begins with the loading of the duplex siRNA by removing the RNA-induced silencing complex (RISC) and the passenger (sense) strand to create a functional RISC containing only the leader (antisense) strand. RISC-loaded guide strands then bind to the complementary target mRNA via Watson-Crick base pairs, triggering endonucleolytic cleavage of the target mRNA opposite the guide nucleotides at positions 10-11 of the 5′ end. After nearly two decades of research and development, ONPATTRO, a partially modified siRNA encapsulated in lipid nanoparticles, became the first RNAi drug to receive marketing approval in 2018.
Acetylgalactosamine (GalNAc) ligand combined with extensive chemical modifications to stabilize siRNA enabled selective targeting of hepatocytes in the liver via the asialoglycoprotein receptor (ASGPR) and has transformed liver-directed therapeutic oligonucleotides (Nair et al., 2014). An early generation of GalNAc-conjugated siRNA using standard template chemistry (STC) with only a few additional modifications achieved clinical proof of concept, but required a high and frequent dosing schedule (Zimmermann et al., 2017). Subsequent design improvements, including replacing the two terminal phosphodiester bonds at the 3′ and 5′ ends of the antisense and the 5′ end of the sense strand with phosphorothioate bonds, led to an improved stabilization chemistry (ESC ) design with improved metabolic stability and potency, allowing for a reduction in the total required dosage and enabling less frequent administration (Nair et al., 2017). GIVLAAR, the first ESC-designed GalNAc-conjugated siRNA, received regulatory approval in 2019. It is dosed at 2.5 mg/kg per month as a subcutaneous injection. Continued refinement of the chemical modification pattern led to the development of improved ESC designs with increased metabolic stability (Foster et al., 2018) and the inclusion of seed destabilizing modifications such as glycol nucleic acid provides improved specificity, called the ESC + design (Janas) . et al., 2018). These advances have led to the expansion of target protein depletion in non-clinical studies and exploratory clinical trials (Ray et al., 2020). Vutrisiran, an advanced ESC GalNAc-conjugated siRNA currently in phase 3 clinical development, shows sustained pharmacodynamic (PD) effects lasting up to 10 months after a single 25 mg s.c. management dose in healthy volunteers (Habtemariam et al., 2021).
Unlike traditional small molecule drugs, siRNAs are large hydrophilic molecules. The two complementary strands of siRNA form double helical structures of 19-21 bp with a molecular weight of ~14,000 kDa. Their polyanionic framework and hydrophilic character prevent passive uptake across cell membranes and therefore require special delivery solutions to achieve adequate cellular uptake. Over the past several years, knowledge of nonclinical ADME properties has been accumulated using dozens of GalNAc-conjugated siRNAs, including STC, ESC, Advanced ESC, and ESC+ designs.
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Data from nonclinical studies of GalNAc-conjugated siRNAs suggest that rapid hepatic uptake is followed by slow hepatic metabolism and clearance after subcutaneous administration ( Nair et al., 2017 ; Zimmermann et al., 2017 ). As such, plasma concentrations are transient and do not directly reflect the long-term duration of PD. Peak plasma concentrations after subcutaneous administration of GalNAc-conjugated siRNA in all nonclinical species were typically reached approximately 0.25 to 2 hours after administration, with the last detectable plasma concentration being the lower limit of quantification observed between 4 and 12 hours, which indicating rapid and efficient hepatic absorption (Nair et al., 2017; Foster et al., 2018). This was reproducible for different siRNA conjugate chemistries and different non-clinical species. In contrast, peak concentrations in mice, rats and monkeys are typically observed 2-8 h after subcutaneous administration, underlining the rapid and efficient absorption of GalNAc-siRNA. Recent work highlights an acidic intracellular depot that significantly contributes to the persistent PD effect (Brown et al., 2020). This work shows that the high metabolic stability of the current generation of GalNAc-conjugated siRNAs in intracellular compartments leads to the continuous and sustained release of functionally active siRNAs into the cytoplasm, where they can be loaded into RISC and a long-lasting PD induce effect.
The current series of non-clinical ADME studies have been conducted over the years to support the continued development of GalNAc-siRNA. The purpose of this work is to summarize the acquired knowledge about absorption, distribution, metabolism and excretion (ADME) properties; compare and contrast molecules that contain different sequences but similar chemical modifications; and to provide a reference to our completed work to describe the translation between non-clinical PK properties and human ones.
Clarity OTX lysis loading buffer and Clarity OTX 96-well solid phase extraction plates were obtained from Phenomenex (Torrance, CA). Optima liquid chromatography-mass spectrometry (LC-MS) grade acetonitrile and water, 6-fold electrophoretic mobility shift assay (EMSA) gel loading solution, and SYBR gold nucleic acid gel stain were purchased from Thermo Scientific (Waltham, MA) and 1 1 , 1 , 3, 3, 3-hexafluoro-2-propranol (HFIPA),
Acetylglucosaminidase, nuclease P1, and glycobuffer 1 were purchased from New England Biolabs. Tris-borate-EDTA (10%) gel was from Bio-Rad Laboratories (CA). Oligonucleotides were synthesized in Alnylam by solid-phase synthesis using an RNA synthesizer. Sterling solvents/reagents from Glen Research, 500-Å controlled-pore glass solid support from Prime Synthesis, and 2′-OMe, 2′-F nucleoside-3′-phosphoramidides from Hongene were used as received. Empty water acetonitrile was purchased from EMD Chemicals. Male hepatocytes were purchased from BioIVT (Westbury, NY). All siRNAs had HPLC purity >90% and all tritium-labeled siRNAs had HPLC radiopurity >96% and tritium-specific activity >250 uCi/mg siRNA. Tritium-specific activity was measured using a Hidex 300 SL liquid scintillation counter (Lablogic).
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Synthesis of GalNAc-siRNAs used in this assay was performed as previously described ( Nair et al., 2014 ). Common chemical modifications previously disclosed for late-stage siRNAs, including revusiran, givosiran, and lumasiran, were used in all GalNAc siRNAs described throughout this report (Shen and Corey, 2018) and are depicted in Figure 1 ; in cases where glycol nucleic acid was used, the design was consistent with that published (Schlegel et al., 2017). To assess sex-dependent differences in GalNAc-siRNA PK, all data obtained from male and female animals were included in the analysis. For mechanistic studies involving in vitro confirmation of duplex metabolism, GalNAc-siRNA was used as a surrogate for siRNA2 because it reflects the ESC design and has a complete data package (including radiolabeled ADME, QWBA, and metabolite- assessment, including metabolite assignment for contribution to pharmacology). All data from the 5/6 nephrectomy model and rat ADME/QWBA studies are included. The siRNA used in these studies was decided based on individual projects and project stage
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