Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is significantly upregulated in glioblastoma (GBM) and plays a crucial role in cell apoptosis and drug resistance. Micheliolide (MCL) is a natural product with a variety of antitumour activities, and the fumarate salt form of dimethylamino MCL (DMAMCL: commercial name ACT001) has been tested in clinical trials for recurrent GBM. Our previous work has revealed that MCL/DMAMCL could suppress the proliferation of GBM cells by rewiring aerobic glycolysis. Herein, we demonstrated that MCL directly targets GAPDH through covalent binding to the cysteine 247 (Cys247) residue. Intriguingly, MCL does not affect the enzymatic activity of GAPDH but facilitates the nuclear translocation of the GAPDH/Siah1 (E3 ligase) complex. Furthermore, MCL/DMAMCL can exacerbate temozolomide (TMZ)-induced DNA damage. This treatment synergistically induced GBM cell death and suppressed tumour growth in a GBM xenograft mouse model. Collectively, our results reveal that MCL triggers non-glycolysis-related functions of GAPDH and that MCL promotes GBM cell death, especially when combined with TMZ, thus providing a novel strategy for clinical GBM treatment.

To ensure specificity, loading of the sense strand of small interfering RNAs (siRNAs) into RISC must be inhibited. We show here that siRNAs with 5 '- and 6 '-morpholino LNA residues or 6 '-OH-LNA at the 5 ' terminus of a fully phosphodiester sense strand resulted in metabolically stable siRNAs with a potency and a duration of action in mice that were greater than those of an siRNA in which the 5 ' terminus of the sense strand has two terminal phosphorothioate linkages and regular LNA.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural β-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and β-anomeric linkages, N-glycosides with β-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N -acetylga-lactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterol-emia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor – mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conju-gated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. (anti-sense single-strand single

acute hepatic porphyria; ALA, delta-aminolevulinic acid; ALAS1 , 5ʹ-aminolevulinate synthase 1; ASGPR; asialglycoprotein receptor; AUC, area under the curve; CL, total clearance; C max , maximum observed concentration; CYP, cytochrome P450; DDI, EDTA, ethylenediaminetetraacetic acid; GalNAc, N-acetylgalactosamine; IV, intravenous; LC-HRMS, liquid chromatography coupled with high resolution mass spectrometry; mRNA, messenger RNA; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PBG, porphobilinogen; PD, pharmacodynamics; P-gp, P-glycoprotein; PK, pharmacokinetics; PPB, plasma protein binding; RISC, RNA-induced silencing complex; RNAi, RNA interference; SC, subcutaneous; SD; standard deviation; siRNA, small interfering RNA; t 1/2 , half-life; V ss , volume of distribution steady Abstract therapeutic that targets 5ʹ-aminolevulinate synthase 1 ( ALAS1 ) messenger RNA (mRNA) in the liver and is currently marketed for the treatment of acute hepatic porphyria (AHP). Herein, nonclinical pharmacokinetic (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of givosiran were characterized. Givosiran was completely absorbed after subcutaneous (SC) administration with relatively short plasma elimination t 1/2 (less than 4 hours). Plasma exposure increased approximately dose proportionally with no accumulation after repeat doses. Plasma protein binding (PPB) was concentration dependent across all species tested and was around 90% at clinically relevant concentration in human. Givosiran predominantly distributed to the liver by asialoglycoprotein receptor (ASGPR)-mediated uptake, and the elimination t 1/2 in the liver was significantly longer (~1 week). Givosiran was metabolized by nucleases, not cytochrome P450 (CYP) isozymes, across species with no human unique metabolites. Givosiran metabolized to form one primary active metabolite with the loss of 1 nucleotide from the 3ʹ end of antisense strand, AS(N-1)3ʹ givosiran which was equipotent to givosiran. Renal and fecal excretion were minor routes of elimination of givosiran as approximately 10% and 16% of the dose was recovered intact in excreta of rats and monkeys, respectively. Givosiran is not a substrate, inhibitor, or inducer of CYP isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, givosiran has a low potential of mediating drug-drug interactions involving CYP isozymes and drug transporters. metabolite

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].

Givosiran is an N-acetylgalactosamine–conjugated RNA interference therapeutic that targets 5′-aminolevulinate synthase 1 mRNA in the liver and is currently marketed for the treatment of acute hepatic porphyria. Herein, nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion properties of givosiran were characterized. Givosiran was completely absorbed after subcutaneous administration with relatively short plasma elimination half-life (t1/2; less than 4 hours). Plasma exposure increased approximately dose proportionally with no accumulation after repeat doses. Plasma protein binding was concentration dependent across all species tested and was around 90% at clinically relevant concentration in human. Givosiran predominantly distributed to the liver by asialoglycoprotein receptor–mediated uptake, and the t1/2 in the liver was significantly longer (∼1 week). Givosiran was metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species with no human unique metabolites. Givosiran metabolized to form one primary active metabolite with the loss of one nucleotide from the 3′ end of antisense strand, AS(N-1)3′ givosiran, which was equipotent to givosiran. Renal and fecal excretion were minor routes of elimination of givosiran as approximately 10% and 16% of the dose was recovered intact in excreta of rats and monkeys, respectively. Givosiran is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, givosiran has a low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT Nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion (ADME) properties of givosiran were characterized. Givosiran shows similar pharmacokinetics and ADME properties across rats and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in adequate exposure of givosiran to the target organ (liver). These studies support the interpretation of toxicology studies, help characterize the disposition of givosiran in humans, and support the clinical use of givosiran for the treatment of acute hepatic porphyria.

Aim: Advancements in RNA interference therapeutics have triggered development of improved bioanalytical methods for oligonucleotide metabolite profiling and high-throughput quantification in biological matrices. Results & methodology: HPLC coupled with high-resolution mass spectrometry (LC-HRMS) methods were developed to investigate the metabolism of a REVERSIR (TM) molecule in vivo. Plasma and tissue samples were extracted using solid-phase extraction followed by LC-HRMS analysis for metabolite profiling and quantification. The method was qualified from 10 to 5000 ng/ml (plasma) and 100 to 50000 ng/g (liver and kidney). In rat liver, intra and interday accuracy ranged from 80.9 to 118.5% and 88.4 to 111.9%, respectively, with acceptable precision (<20% CV). Conclusion: The LC-HRMS method can be applied for metabolite profiling and quantification of oligonucleotides in biological matrices.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2-deoxy-2-fluoro (2-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2-F-monomer metabolites was low and transient in rats and humans. In vitro, 2-F-nucleoside 5-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for approximate to 2 years. Taken together, the results support the conclusion that 2-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Aim: A novel single-stranded deaminated oligonucleotide metabolite resulting from a REVERSIR™ oligonucleotide was discovered and identified in monkey liver after subcutaneous administration. Results & methodology: REVERSIR-A and its metabolites were extracted from biological matrices by solid phase extraction and analyzed using LC coupled with high-resolution MS under negative ionization mode. A novel 9-mer metabolite of REVERSIR-A, resulting from deamination of the 3' terminal 2'-O-methyl-adenosine nucleotide to 2'-O-methyl-inosine, was discovered at significant levels in monkey liver. The metabolite's identity was confirmed by LC-MS/MS. Conclusion: This report describes the first observation of a long-chain deaminated metabolite of a single-stranded REVERSIR oligonucleotide in vivo in monkey liver.

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5 '-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5 '-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into ki-netics of RISC loading and demonstrated excellent translation to non-human primates.

Registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-O-methyl ribose modifications of multiple nucleotides in the ICH battery of assays, all of which have uniformly yielded negative results. To verify these negative genotoxicity results, in this study we confirm test article exposure using toolkit small interfering RNAs (siRNAs) representative of those in the clinic. In the Ames test, the highest uptake of the siRNA-GalNAc conjugates occurred at 1 h postdose in all bacterial strains independent of siRNA sequence or chemistry (up to ∼14,000 siRNA molecules per cell), followed by metabolic degradation of the parent siRNA at 6, 24, and 48 h postdose. siRNA-GalNAc conjugates were internalized by bacteria as assessed by protection from the addition of nucleases to the culture media following uptake and by the requirement of cell lysis for detection of the siRNA. In the in vitro chromosome aberration assay, uptake was observed in Chinese hamster ovary cells (up to ∼5,500 siRNA molecules per cell at 21 h postdose) and in CD3+ human peripheral blood lymphocytes (up to ∼500 siRNA molecules per cell at 21 h postdose). In the in vivo micronucleus assay in rat bone marrow, exposure to parent siRNA was 100-350 μg of antisense strand per gram of protein at 24 and 48 h postlimit dose of 2 g/kg. Loss of terminal nucleotides was detected in bone marrow by mass spectrometry, indicating exposure to monomer metabolites as well. Negative genotoxicity results were also confirmed in an in vitro double-strand DNA break assay in HeLa and HepG2 cells where exposure was maximized using transfection reagents. Thus negative genotoxicity assay results for siRNA-GalNAc conjugates were valid and not the result of poor or no intracellular exposure.

In recent years, RNA interference (RNAi) therapeutics, most notably with lipid nanoparticle-based delivery systems, have advanced into human clinical trials. The results from these early clinical trials suggest that lipid nanoparticles (LNPs), and the novel ionizable lipids that comprise them, will be important materials in this emerging field of medicine. A persistent theme in the use of materials for biomedical applications has been the incorporation of biodegradability as a means to improve biocompatibility and/or to facilitate elimination. Therefore, the aim of this work was to further advance the LNP platform through the development of novel, next-generation lipids that combine the excellent potency of the most advanced lipids currently available with biodegradable functionality. As a representative example of this novel class of biodegradable lipids, the lipid evaluated in this work displays rapid elimination from plasma and tissues, substantially improved tolerability in preclinical studies, while maintaining in vivo potency on par with that of the most advanced lipids currently available.

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8(+) T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.