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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.

The efficient resolution of racemic N-benzyl α-amino acids (N-Bn-AA) has been achieved by a liquid-liquid extraction process using the lipophilic chiral salen–cobalt(III) complex [CoIII(3)(OAc)]. As a result of the resolution by extraction, one enantiomer (S) of the N-benzyl α-amino acid predominated in the aqueous phase, while the other enantiomer (R) was driven into the organic phase by complexation to cobalt. The complexed amino acid (R) was then quantitatively released by a reductive (CoIII → CoII) counter-extraction with aqueous sodium dithionite or L-ascorbic acid in methanol. Thereductive cleavage allowed to recover the [CoII(3)] complex in good yield, which could be easily re-oxidized to[CoIII(3)(OAc)] with air/AcOH and reused with essentially no loss of reactivity and selectivity. Investigation on the nitrogen substitution indicates that the presence of a single benzyl group on the amino acid nitrogen is important to obtain high enantioselectivity in the extraction process. The kinetic vs. thermodynamic nature of the resolution process was also investigated with an enantiomeric exchange experiment, which shows that the liquid-liquid extraction with [CoIII(3)(OAc)] is an equilibrium process operating under thermodynamic control. In the absence of a suitable crystal structure of the [CoIII(3)(N-Bn-AA)] complexes, computational and spectroscopic studies were used to investigate how the N-benzyl α-amino acids are accommodated in the “binding pocket” of the chiral cobalt complex. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Tissue analysis has always been a difficult discipline of bioanalysis. Differences in scientific approaches or level of adherence to regulated guidelines have led to a growing ambiguity on how to perform tissue analysis, an ambiguity that starts with the question of if we analyze tissue or tissue homogenates. The European Bioanalysis Forum (EBF) is proposing a recommendation on how to perform method establishment and analysis of tissue homogenates. The recommendation is based on broad discussions and survey data from the EBF community and, as for many EBF recommendations, focuses on finding the right balance between science, technology and regulations.

Tissue analysis has always been a difficult discipline of bioanalysis. Laboratories that perform bioanalysis in tissue are facing a lot of challenges and questions before starting experiments, from a scientific/technical point of view regarding more regulated aspects. Actually, literature is poor regarding the more technical and scientific aspects but also beyond that no clear guidance is available on this topic and laboratories performing tissue analysis face real ambiguity regarding regulatory requirements, always with the risk of under- or over-validation of the assay. For all of these reasons bioanalysis in tissue became a frequently discussed topic within the European Bioanalytical Forum (EBF) organization. The EBF then decided to treat this as a specific topic, and carried out a survey that was done in two steps between 2012 and 2013. This paper represents an exhaustive summary of the result of this survey that includes themost important aspects of tissue bioanalysis. This survey provided the team a good starting point for their discussions and resulted in an EBF recommendation paper published separately.

The efficient (up to 93% ee) resolution of racemic N-benzyl beta(3)-amino acids has been achieved by an iterative (two cycle) liquid-liquid extraction process using a lipophilic chiral (salen)cobalt(III) complex [Co(III)(1)(OAc)]. As a result of the resolution by extraction, one enantiomer of the N-benzyl beta(3)-amino acid predominated in the aqueous phase, while the other enantiomer was driven into the organic phase by complexation to cobalt. The complexed amino acid was then quantitatively released into an aqueous phase, by a reductive (Co(III)--Co(II)) counter-extraction using l-ascorbic acid. The reductive cleavage allowed for the recovery of the cobalt(II) selector in up to 90% yield (easily re-oxidable to Co(III) with air/AcOH).