Based on a novel method for solid-phase extraction using cationic polystyrene nanoparticles, the suitability of the extraction procedure for quantitation of terminally and backbone-modified antisense oligonucleotides was investigated. Extractions were carried out from both human plasma and urine. Quantitative analysis of the extracted samples was performed with capillary gel electrophoresis. In accordance with previous results obtained with phosphorothioate oligonucleotides in human plasma, high linearity and accuracy of the assay was demonstrated for an oligodeoxyribonucleotide-palmityl conjugate as well as for a modified oligoribonucleotide. Optimized extraction conditions allow the isolation of oligonucleotides in high yields and purity even for concentrations in the low nanomolar range, down to 5 nM. Comparing the results obtained from human plasma and urine, no significant differences in the absolute recovery rates which reach values up to 95% were observed. However, when the loading capacity of the nanoparticles was exceeded, selective recovery was observed for the coisolation of phosphodiester and phosphorothioate oligonucleotides. This effect can be explained by differences in the attractive forces between PO-and PS-oligonucleotides and the particle surface and appears to be valuable for a modification-dependent enrichment of oligonucleotides out of complex mixtures. © 1998
Academic Press
Antisense oligonucleotides (ONs) 2 offer the potential to inhibit gene expression by specific binding to a target sequence located on the mRNA, hence preventing the translation process (1, 2). In contrast to common pharmaceutical drug development, antisense compounds can rationally be designed against any protein as far as the sequence of the corresponding gene is known. Natural phosphodiester ONs, however, suffer from poor transport through cellular membranes and are rapidly degraded by intra-and extracellular nucleases (3,4). A large number of chemical modifications have been introduced to overcome these stability problems and to improve the delivery and cellular uptake of the antisense compounds [for reviews see: Refs. (5-7)]. However, progress in the areas of sequence specificity and potency of the agents has only recently been addressed. Promising results were obtained with novel derivatives such as oligonucleotidelipid conjugates, ONs with modified nucleobases, and mixed backbone ONs composed of modified .
A number of these so-called antisense ONs of the second-generation are currently undergoing preclinical or clinical investigations (12, 13). Thus, reliable analytical techniques are required for their quantitation in biological fluids, such as human plasma, urine, cellular extracts, and tissue homogenates, in order to make studies of the pharmacokinetics and metabolism feasible.
In a previously published work a novel method for solid-phase extraction of phosphorothioate oligonucleotides (PTOs) from human plasma has been developed 1 To whom correspondence should be addressed at Forschungsstelle fu ¨r Nucleinsa ¨ure-und Peptidchemie,