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Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

A novel approach to tritium-labeled antisense oligonucleotides (ASO) was established by conjugating N-succinimidyl propionate, as well as maleimide-derivatives, to the 3′-end of ASOs targeting metastasis-associated lung adenocarcinoma transcript 1 (Malat1) containing amino- or sulfhydryl-linkers. In vitro stability and Malat1 RNA reduction studies demonstrated that N-ethylmaleimide (NEM) could be used as a stable tag while maintaining the desired target interaction. The corresponding radioactive label conjugation using [3H]-NEM resulted in tritium-labeled ASOs with a high molar specific activity of up to 17 Ci/mmol. Single-dose in vivo studies in mice were carried out to compare [3H]-ASOs with their unlabeled counterpart ASOs, with and without conjugation to N-acetylgalactosamine (GalNAc), for tissue and plasma concentrations time profiles. Despite the structural modification of the labeled ASOs, in vitro target interaction and in vivo pharmacokinetic behaviors were similar to that of the unlabeled ASOs. In conclusion, this new method provides a powerful technique for fast and safe access to tritium-labeled oligonucleotides, e.g., for pharmacokinetic, mass balance, or autoradiography studies. Graphical abstract

An assay was developed and applied to determine the bioavailability of a new endothelin receptor antagonist after simultaneous p.o. and i.v. administration of the drug and its stable isotope-labeled analogue. The drug, its main metabolite and the stable isotope-labeled analogues of the drug and the main metabolite were quantified in dog plasma samples using a structural analogue as internal standard. In addition to the calculation of the bioavailability, the formation of the metabolite after p.o. and i.v. administration could be followed independently. The assay covered the concentration range 0.25-1000 ng ml(-1) using sample aliquots of only 50 micro l. Plasma samples were processed after protein precipitation with on-line solid-phase extraction, narrow-bore high-performance liquid chromatography and subsequent tandem mass spectrometric detection. Detection was accomplished with ionspray in the positive ion selected reaction monitoring mode. The inter-assay precision and accuracy of the assay were in the range 4.7-14.2% and 90.3-113.3%, respectively, and the intra-assay precision and accuracy were in the range 1.4-11.5% and 88.4-112.5%, respectively. The fragmentation of the drug was investigated and showed an unexpected shift of a methyl group. Data from MS(n), medium-resolution exact mass tandem mass spectrometry and H-D exchange experiments were employed to clarify the mechanism.

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